Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines

ABSTRACT

Novel oligomers are disclosed which have enhanced ability with respect to forming duplexes or triplexes compared with oligomers containing only conventional bases. The oligomers contain the bases 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine or related analogs. The oligomers of the invention are capable of (i) forming triplexes with various target sequences such as virus or oncogene sequences by coupling into the major groove of a target DNA duplex at physiological pH or (ii) forming duplexes by binding to single-stranded DNA or to RNA encoded by target genes. The oligomers of the invention can be incorporated into pharmaceutically acceptable carriers and can be constructed to have any desired sequence, provided the sequence normally includes one or more bases that is replaced with the analogs of the invention. Compositions of the invention can be used as pharmaceutical agents to treat various diseases such as those caused by viruses and can be used for diagnostic purposes in order to detect viruses or disease conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 08/599,738,filed on Feb. 12, 1996, now U.S. Pat. No. 6,380,368, which is adivisional of application Ser. No. 07/976,103, filed on Nov. 25, 1992,now U.S. Pat. No. 5,645,985, and a continuation of application Ser. No.08/338,352, filed on Nov. 14, 1994, now U.S. Pat. No. 6,235,887, whichis a continuation-in-part of application Ser. No. 07/935,444, filed Aug.25, 1992, now abandoned. Application Ser. No. 07/976,103 is acontinuation-in-part of application Ser. No. 07/965,941, filed on Oct.23, 1992, now abandoned, which is a continuation-in-part of applicationSer. No. 07/935,444. Application Ser. No. 07/935,444, in turn, is acontinuation-in-part of application Ser. No. 07/799,824, filed Nov. 26,1991, now U.S. Pat. No. 5,484,908.

TECHNICAL FIELD

The invention relates generally to novel nucleomonomer and oligomeranalogs, and to oligonucleotide-based therapy and diagnosis by bindingof the oligonucleotide analogs to single or double-stranded nucleic acidtarget sequences. More specifically, the invention concerns oligomerscontaining certain 5-substituted pyrimidine base residues andintermediates in their synthesis.

BACKGROUND ART

Sequence-specific binding of oligonucleotides both to single-strandedRNA and DNA and to duplex DNA has been demonstrated. The appropriatesequence recognition for binding to single-stranded targets is wellknown: the A-T and G-C pairing characteristic of duplex formation hasbeen established as the basis for DNA replication and transcription.

More recently, oligonucleotides have been shown to bind in asequence-specific manner to duplex DNA to form triplexes.Single-stranded nucleic acid, primarily RNA, is the target molecule foroligonucleotides that are used to inhibit gene expression by an“antisense” mechanism (Uhlmann, E., et al, Chem Reviews (1990) 90:543-584; van der Krol, A. R., et al, Biotechniques (1988) δ: 958-976).Antisense oligonucleotides are postulated to exert an effect on targetgene expression by hybridizing with a complementary RNA sequence. Inthis model, the hybrid RNA-oligonucleotide duplex interferes with one ormore aspects of RNA metabolism including processing, translation andmetabolic turnover. Chemically modified oligonucleotides have been usedto enhance their nuclease stability.

Duplex DNA can be specifically recognized by oligomers based on arecognizable nucleomonomer sequence. Two major recognition motifs havebeen recognized. In an earlier description of a “CT” motif, protonatedcytosine residues recognize G-C basepairs while thymine residuesrecognize A-T basepairs in the duplex. These recognition rules areoutlined by Maher III, L. J., et al., Science (1989) 245: 725-730;Moser, H. E., et al., Science (1987) 238: 645-650. More recently, anadditional motif, termed “GT” recognition, has been described (Beal, P.A., et al, Science (1992) 251: 1360-13.63; Cooney, M., et al., Science(1988) 241: 456-459; Hogan, M. E., et al., EP Publication 375408). Inthe G-T motif, A-T pairs are recognized by adenine or thymine residuesand G-C pairs by guanine residues.

In both of these binding motifs, the recognition sequence of theoligomer must align with the complementary sequence of the purine chainof the duplex; thus, recognition, for example, of an A-T pair by athymine, depends on the location of the adenyl residues along the purinechain of the duplex. An exception to the foregoing is the recent reportby Griffin, L. C., et al., Science (1989) 245: 967-971, that limitednumbers of guanine residues can be provided within pyrimidine-richoligomers and specifically recognize thymine-adenine base pairs; thispermits the inclusion of at least a limited number of pyrimidineresidues in the homopurine target.

The two motifs exhibit opposite binding orientations with regard tohomopurine target chains in the duplex. In the CT motif, the targetingoligonucleotide is oriented parallel to the target purine-rich sequence;in the GT motif, the oligonucleotide is oriented antiparallel (Beal, P.A., et al., Science (1990) 251: 1360-1363).

The efficiency of binding by C residues in CT motif oligomers is reducedas the pH of hybridization is increased. The protonated tautomer ofC(C⁺) is the binding competent species in Hoogsteen binding, but ispresent at only low levels at physiological pH. This is consonant withthe pK_(a) of cytosine which is 4.25. Base analogs such as5-methylcytosine, pK_(a) 4.35, (Lee, J. S. et al., Nucleic Acids Res(1984) 12: 6603-6614), 8-oxo-N⁶-methyladenine (Krawczyk, S. H. et al,Proc Natl Acad Sci (1992) 89: 3761-3764; International Application No.PCT/US91/08811), pseudoisocytidine (Ono, A., et al, J Org Chem (1992)57: 3225-3230; International Application No. PCT/US90/03275) orcarbocyclic cytidine (Froehler, B. C., et al, J Am Chem Soc (1992) 114:8320-8322; U.S. patent application Ser. No. 07/864,873 incorporatedherein by reference) have been utilized to obtain binding of CT motifoligomers over an extended pH range.

Sequence-specific targeting of both single-stranded and duplex targetsequences has applications in diagnosis, analysis, and therapy. Undersome circumstances wherein such binding is to be effected, it isadvantageous to stabilize the resulting duplex or triplex over long timeperiods.

Covalent crosslinking of the oligomer to the target provides oneapproach to prolong stabilization. Sequence-specific recognition ofsingle-stranded DNA accompanied by covalent crosslinking has beenreported by several groups. For example, Vlassov, V. V., et al., NucleicAcids Res (1986) 14: 4065-4076, describe covalent bonding of asingle-stranded DNA fragment with alkylating derivatives ofnucleomonomers complementary to target sequences. A report of similarwork by the same group is that by Knorre, D. G., et al., Biochimie(1985) §67: 785-789. Iverson and Dervan also showed sequence-specificcleavage of single-stranded DNA mediated by incorporation of a modifiednucleomonomer which was capable of activating cleavage (J Am Chem Soc(1987) 109: 1241-1243). Meyer, R. B., et al., J Am Chem Soc (1989) 111:8517-8519, effect covalent crosslinking to a target nucleomonomer usingan alkylating agent complementary to the single-stranded targetnucleomonomer sequence. Photoactivated crosslinking to single-strandedoligonucleotides mediated by psoralen was disclosed by Lee, B. L., etal., Biochemistry (1988) 27: 3197-3203. Use of crosslinking intriple-helix forming probes was also disclosed by Horne, et al., J AmChem Soc (1990) 112: 2435-2437.

Use of N⁴,N⁴-ethanocytosine as an alkylating agent to crosslink tosingle-stranded and double-stranded oligomers has also been described(Webb and Matteucci, J Am Chem Soc (1986) 108: 2764-2765; Nucleic AcidsRes (1986) 14: 7661-7674; Shaw, J. P., et al, J Am Chem Soc (1991) 113:7765-7766). These papers also describe the synthesis of oligonucleotidescontaining the derivatized cytosine. Matteucci and Webb, in a laterarticle in Tetrahedron Letters (1987) 28: 2469-2472, describe thesynthesis of oligomers containing N⁶,N⁶-ethanoadenine and thecrosslinking properties of this residue in the context of anoligonucleotide binding to a single-stranded DNA.

In a recent paper, Praseuth, D., et al., Proc Natl Acad Sci (USA) (1988)85: 1349-1353, described sequence-specific binding of an octathymidylateconjugated to a photoactivatable crosslinking agent to bothsingle-stranded and double-stranded DNA.

In addition, Vlassov, V. V. et al., Gene (1988) 313-322 and Fedorova, O.S. et al., FEBS (1988) 228: 273-276, describe targeting duplex DNA withan alkylating agent linked through a 5′-phosphate of an oligonucleotide.

In effecting binding to obtain a triplex, to provide for instanceswherein purine residues are concentrated on one chain of the target andthen on the opposite chain, oligomers of inverted polarity can beprovided. By “inverted polarity” is meant that the oligomer containstandem sequences which have opposite polarity, i.e., one having polarity5′→3′ followed by another with polarity 3′→5′, or vice versa. Thisimplies that these sequences are joined by linkages which can be thoughtof as effectively a 3′-3′ internucleoside junction (however the linkageis accomplished), or effectively a 5′-5′ internucleoside junction. Sucholigomers have been suggested as by-products of reactions to obtaincyclic oligonucleotides by Capobionco, M. L., et al., Nucleic Acids Res(1990) 18: 2661-2669. Compositions of “parallel-stranded DNA” designedto form hairpins secured with AT linkages using either a 3′-3′ inversionor a 5′-5′ inversion have been synthesized by van de Sande, J. H., etal., Science (1988) 241: 551-557. In addition, triple helix formationusing oligomers which contain 3′-3′ linkages have been described (Horne,D. A., and Dervan, P. B., J Am Chem Soc (1990) 112: 2435-2437; Froehler,B. C., et al, Biochemistry (1992) 31: 1603-1609).

The use of triple helix (or triplex) complexes as a means for inhibitionof the expression of target gene expression has been previously adduced(International Application No. PCT/US89/05769). Triple helix structureshave been shown to interfere with target gene expression (InternationalApplication No. PCT/US91/09321; Young, S. L. et al, Proc Natl Acad Sci(1991) 88: 10023-10026), demonstrating the feasibility of this approach.

European Patent Application No. 92103712.3, Rahim, S. G., et al(Antiviral Chem Chemother (1992) 3: 293-297), and InternationalApplication No. PCT/SE91/00653 describe pyrimidine nucleomonomercharacterized by the presence of an unsaturated group in the 5-position.Propynyl and ethynyl groups are included among the derivatives at the5-position that are described in the applications.

Synthesis of nucleomonomers having unsaturated alkyl groups at the5-position of uracil has been described (DeClercq, E., et al, J Med Chem(1983) 26: 661 -666; Goodchild, J., et al, J Med Chem (1983) 26:1252-1257). Oligomers containing 5-propynyl modified pyrimidines havebeen described (Froehler, B. C., et al, Tetrahedron Letters (1992) 33:5307-5310).

Conversion of 5-propynyl-2′-deoxyuridine, 5-butynyl-2′-deoxyuridine andrelated compounds to the 5′-triphosphate followed by incorporation ofthe monomer into oligomers by E. coli polymerase has been described(Valko, K., et al, J Liquid Chromatoq (1989) 12: 2103-2116; Valko, K. etal, J Chromatoq (1990) 506: 35-44). These studies were conducted as astructure to activity analysis of nucleotide analogs having a series ofsubstitutions at the 5position of uracil. The activity of the nucleotideanalogs as substrates for E. coli polymerase was examined and correlatedwith characteristics such as the hydrophobicity of the monomer. Noinformation was presented regarding the properties of oligomerscontaining the analogs.

European patent application 0492570 published Jul. 1, 1992 describes amethod for detecting a target polynucleotide using a single-strandedpolynucleotide probe in which an intercalating molecule is attached by alinker which comprises at least 3 carbon atoms and a double bond at thealpha position relative to the base.

PCT patent publication WO 92/02258 describes nuclease resistant,pyrimidine modified oligomers including a substituent group at the 5 or6 positions, including phenyl. 5-Phenyl-2′-deoxyuridine has beensubsequently incorporated into oligomers and shown to decrease thebinding affinity of oligomers containing this modification for bothsingle stranded and double stranded target sequences.

DNA synthesis via amidite and hydrogen phosphonate chemistries has beendescribed (U.S. Pat. Nos. 4,725,677; 4,415,732; 4,458,066; 4,959,463).

Oligomers having enhanced affinity for complementary target nucleic acidsequences would have improved properties for diagnostic applications,therapeutic applications and research reagents. Thus, a need exists foroligomers with enhanced binding affinity for complementary sequences.Oligomers of the present invention have improved binding affinity fordouble stranded and/or single stranded target sequences.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Dimer synthons containing bases of the invention.

FIG. 2. Dimer synthons containing bases of the invention and containing5 and 6 membered riboacetal type linkages.

FIG. 3. Dimer synthons containing bases of the invention and containing6 and 7 membered riboacetal type linkages.

FIGS. 4-1 and 4-2. Synthesis of monomers containing 2′-O-allylmodifications.

FIGS. 5-1 and 5-2. Synthesis of O-xylene linked switchback dimers(synthetic method #1).

FIG. 6. Synthesis of monomers containing 2′-S-alkyl modifications.

FIG. 7. Synthesis of dimer linked by a 3′-thioformacetal linkage (method#2).

FIGS. 8-1 and 8-2. Synthesis of trimer linked by a 3′-thioformacetallinkage (method #2).

FIGS. 9-1 and 9-2. Synthesis of dimer linked by a riboacetal linkage(method #3).

FIGS. 10-1, 10-2, 10-3 and 10-4. Coupling groups for oligomer synthesisvia amidite or triester chemistry.

FIG. 11. Synthesis of dimer linked by a formacetal linkage (method #2).

FIGS. 12-1, 12-2 and 12-3. Oligomer synthesis by (1)hydrogen-phosphonate, (2) amidite chemistry and (3) methyl phosphonatederivatives (method #1).

FIG. 13. Synthesis of a monomer for an oligomer containing amidelinkages (method #4).

FIG. 14. Synthesis of the 5-((1-ethynyl)-2pyrimidinyl)-2′-deoxyuridinenucleomonomer.

FIG. 15. Synthesis of 5-(2-pyridinyl)-2′-deoxyuridine and5-(2-pyridinyl)-2′-deoxycytidine nucleomonomers.

FIG. 16. Synthesis of 5-(2-thienyl)-2′-deoxyuridine derivative.

FIG. 17-1 Series of repeating nucleomonomer units.

FIGS. 17-2 and 17-3. Exemplary amide-linked oligomer structurescontaining selected repeating units having base analogs of theinvention.

FIG. 18. Dimer synthons containing bases of the invention and havingexemplary 2′, 5′ linkages; thioformacetal and carbamate linkages.

STRUCTURAL FORMULAS

Structural formulas that are described herein are designated as anumeral in parentheses ((1), (2), etc.) and chemical compounds aredesignated as an underlined numeral (1, 2, etc.).

DISCLOSURE OF THE INVENTION

The invention provides an oligomer comprising at least two andpreferably a multiplicity of nucleomonomers wherein at least onenucleomonomer comprises a base of formula (1) or (2)

wherein each X is independently O or S;

-   -   R² is a group comprising at least one pi bond connected to a        carbon atom attached to the base; and    -   Pr is (H)₂ or a protecting group,    -   with the proviso that when at least one of said nucleomonomers        of said oligomer comprises deoxyuridine 5-substituted by vinyl,        1-butenyl, 1-pentenyl, 1-hexenyl, 1-heptenyl, 1-octenyl,        1-propynyl, 1-butynyl, 1-hexynyl, 1-heptynyl, or 1-octynyl, then        the remainder of the nucleomonomers comprising said oligomer are        not solely comprised of phosphodiester linked 2′-deoxyadenosine,        2′-deoxyguanosine, 2′-deoxycytidine, thymidine or a combination        thereof.        Definitions

The following definitions are brief synopses of terms that are morefully defined hereinbelow.

Nucleomonomer. As used herein, the term “nucleomonomer” means a moietycomprising (1) a base covalently linked to (2) a second moiety.Nucleomonomers include nucleosides and nucleotides. Nucleomonomers canbe linked to form oligomers that bind to target or complementary basesequences in nucleic acids in a sequence specific manner.

A “second moiety” as used herein includes those species which containmodifications of the sugar moiety, for example, wherein one or more ofthe hydroxyl groups are replaced with a halogen, a heteroatom, analiphatic groups, or are functionalized as ethers, amines, and the like.The pentose moiety can be replaced by a hexose or an alternate structuresuch as a cyclopentane ring, a 6-member morpholino ring and the like.Nucleosides as defined herein are also intended to include a base linkedto an amino acid and/or an amino acid analog having a free carboxylgroup and/or a free amino group and/or protected forms thereof.

Base. “Base” as used herein includes those moieties which contain notonly the known purine and pyrimidine heterocycles and the inventionpyrimidines, but also heterocycle analogs and tautomers thereof. Purinesinclude adenine, guanine and xanthine and exemplary purine analogsinclude 8-oxo-N⁶-methyladenine and 7-deazaxanthine. Pyrimidines includeuracil and cytosine and their analogs such as 5-methylcytosine,5-methyluracil and 4,4-ethanocytosine. Invention bases are pyrimidinesderivatized at the 5position. The derivatives are 1-alkenyl-,1-alkynyl-, heteroaromatic- and 1-alkynyl-heteroaromatic modifications.“1-Alkenyl” means an olefinically-unsaturated (double bond containing)acyclic group. “1-Alkynyl” means an acetylenically-unsaturated (triplebond containing) acylic group. “Heteroaromatic” means a compound havingat least one heterocyclic ring, 5 or 6 ring atoms, having physical andchemical properties resembling compounds such as an aromatic group.“Heteroaromatic” also means systems having one or more rings, includingbicyclic moieties such as benzimidazole, benzotriazole, benzoxazole, andindole. A base also includes heterocycles such as 2-aminopyridine andtriazines. 1-Alkynyl-heteroaromatic means 1-ethynyl-heteroaryl whereinheteroaryl is as defined above.

Nucleoside. As used herein, “nucleoside” means a base covalentlyattached to a sugar or sugar analog and which may contain a phosphite orphosphine. The term nucleoside includes ribonucleosides,deoxyribonucleosides, or any other nucleoside which is an N-glycoside orC-glycoside of a base. The stereochemistry of the sugar carbons can beother than that of D-ribose.

Nucleosides include those species which contain modifications of thesugar moiety, for example, wherein one or more of the hydroxyl groupsare replaced with a halogen, a heteroatom, an aliphatic groups, or arefunctionalized as ethers, amines, thiols, and the like. The pentosemoiety can be replaced by a hexose or an alternate structure such as acyclopentane ring, a 6-member morpholino ring and the like. Nucleosidesas defined herein are also intended to include a base linked to an aminoacid and/or an amino acid analog having a free carboxyl group and/or afree amino group and/or protected forms thereof.

Nucleotide. As used herein, “nucleotide” means nucleoside having aphosphate group or phosphate analog.

Sugar Modification. As used herein, “sugar modification” means anypentose or hexose moiety other than 2′-deoxyribose. Modified sugarsinclude D-ribose, 2′-O-alkyl, 2′-amino, 2′-halo functionalized pentoses,hexoses and the like. Sugars having a stereochemistry other than that ofa D-ribose are also included.

Linkage. As used herein, “linkage” means a phosphodiester moiety(—O—P(O)(O)—O—) that covalently couples adjacent nucleomonomers.

Substitute Linkages. As used herein, “substitute linkage” means anyanalog of the native phosphodiester group that covalently couplesadjacent nucleomonomers. Substitute linkages include phosphodiesteranalogs, e.g. such as phosphorothioate and methylphosphonate, andnonphosphorus containing linkages, e.g. such as acetals and amides.

Switchback. As used herein, “switchback” means an oligomer having atleast one region of inverted polarity. Switchback oligomers are able tobind to opposite strands of a duplex to form a triplex on both strandsof the duplex. The linker joining the regions of inverted polarity is asubstitute linkage.

Oligomers. Oligomers are defined herein as two or more nucleomonomerscovalently coupled to each other by a linkage or substitute linkagemoiety. Thus, an oligomer can have as few as two nucleomonomers (adimer). Oligomers can be binding competent and, thus, can base pair withcognate single-stranded or double-stranded nucleic acid sequences.Oligomers (e.g. dimers-hexamers) are also useful as synthons for longeroligomers as described herein. Oligomers can also contain abasic sitesand pseudonucleosides.

Blocking Groups. As used herein, “blocking group” refers to asubstituent other than H that is conventionally attached to oligomers ornucleomonomers, either as a protecting group, a coupling group forsynthesis, PO₃ ⁻², or other conventional conjugate such as a solidsupport. As used herein, “blocking group” is not intended to beconstrued solely as a protecting group, according to slang terminology,but also includes, for example, coupling groups such as a hydrogenphosphonate or a phosphoramidite.

Protecting group. “Protecting group” as used herein means any groupcapable of preventing the O-atom or N-atom to which it is attached fromparticipating in a reaction or bonding. Such protecting groups for O-and N-atoms in nucleomonomers are described and methods for theirintroduction are conventionally known in the art. Protecting groups alsoprevent reactions and bonding at carboxylic acids, thiols and the like.

Coupling group. “Coupling group” as used herein means any group suitablefor generating a linkage or substitute linkage between nucleomonomerssuch as a hydrogen phosphonate and a phosphoramidite.

Conjugate. “Conjugate” as used herein means any group attached to theoligomer at a terminal end or within the oligomer itself. Conjugatesinclude solid supports, such as silica gel, controlled pore glass andpolystyrene; labels, such as fluorescent, chemiluminescent, radioactive,enzymatic moieties and reporter groups; oligomer transport agents, suchas polycations, serum proteins and glycoproteins and polymers and thelike.

Pi bond. “Pi bond” as used herein means an unsaturated covalent bondsuch as a double or triple bond. Both atoms can be carbon or one can becarbon and the other nitrogen, for example, phenyl, propynyl, cyano andthe like.

Synthon. “Synthon” as used herein means a structural unit within amolecule that can be formed and/or assembled by known or conceivablesynthetic operations.

Transfection. “Transfection” as used herein refers to any method that issuitable for enhanced delivery of oligomers into cells.

Subject. “Subject” as used herein means an animal, including a mammal,particularly a human.

DESCRIPTION OF THE INVENTION

Oligomers including either or both of the modified bases (1) or (2) showenhanced binding capacities in the formation of duplexes or triplexeswith single-stranded RNA or DNA or duplex target sequences,respectively.

When the certain 5-substituted pyrimidines noted above are present, theadditional nucleomonomer modifications can vary widely as discussedhereinafter. Preferably, the additional modification is at least onesubstitute linkage or a sugar modification such as a 2′-substituteddeoxyribose.

The substitution of a base (1) or (2) of the invention, such as in5-(1-alkenyl)-, 5-(1-alkynyl)-, 5-heteroaromatic- or1-alkynyl-heteroaromatic substituted bases for thymine or cytosine inoligomers which target DNA duplexes provides binding competent oligomerswith enhanced binding affinity. Substitution for thymine base residuesby the 5-substituted uracil or thiouracil base residues of the inventionor substitution for cytosine or 2-thiocytosine base residues by the5-substituted cytosine base residues of the invention enhance theability of the resulting oligomer to bind single-stranded DNA or RNAtargets. In addition, some of the 5-substituted pyrimidine base residuessignificantly enhance triple helix formation with double stranded DNA.

For some R², substitution of 5-R² substituted U (5-R²-U) for T inoligomers results in enhanced ability to form triplexes and duplexes ascompared with the oligomers containing thymine. 5-R²-U in theseoligomers, in triplex formation recognizes adenine residues inadenine-thymine base pairs when hybridized in the parallel CT triplexmotif. Oligomers having 8-oxo-N⁶-methyladenine (a cytosine analog fortriplex binding) and 5-R²-U also bind in the CT motif. Oligomers having5-R²-U and guanine are suitable for triplex binding to duplex sequencesvia the GT motif (5-R²-U recognizes adenine). Some oligomers containingsubstitution of 5-R² substituted C (5-R²—C) in place of C bind duplexDNA, but not as well as control oligomers containing 5-methylcytosine atcorresponding positions. The reduced efficiency of triplex formation isbelieved to result primarily from the reduced pK_(a) of the substitutedbase. In the 5-propynyl-substituted nucleomonomer corresponding to thenucleomonomer containing 5-methylcytosine, the pK_(a) is only 3.30.

The oligomers of the invention are thus capable of forming triplexeswith various target sequences such as those found in oncogenes orviruses by binding in the major groove of a target DNA duplex underphysiological pH conditions.

However, alteration of the heterocycle pK_(a) as described above for the5-R²—C does not significantly affect binding to single-stranded targetnucleic acid. In addition to binding efficiently to double-strandedtarget sequences, oligomers of the invention containing 5-R² substitutedU in place of T and/or 5-R² substituted C in place of C were also foundto bind single-stranded RNA efficiently. Oligomers containing either5-R²—C or 5-R²-U formed duplex structures with complementarysingle-stranded RNA that had increased thermal stability (T_(m))compared to the duplex formed by a control oligomer as described below.

Accordingly, in one aspect, the invention is directed to an oligomercomprising at least two and preferably, a multiplicity, ofnucleomonomers wherein at least one said nucleomonomer comprises a baseof formula (1) or (2) above.

Preferably, each X is O, i.e. formula (1) is uracil and formula (2) iscytosine. Other suitable pyrimidines include 4-thiouracil, 2-thiouracil,2,4-dithiouracil and 2-thiocytosine.

In one embodiment of the invention R² is cyano, C₂₋₁₂ 1-alkenyl or1-alkynyl or is a C₂₋₁₂ heteroaromatic group containing 5-6 ring atomsin which one to three of the ring atoms is N, S or O. Preferably, R² isa C₂₋₈ 1-alkenyl or 1-alkynyl or a C₂₋₈ heteroaromatic group containing5-6 ring atoms in which one ring atom is N and optionally a second ringatom is N, S or 0.

By “1-alkenyl” is meant an olefinically-unsaturated acyclic group, forexample, vinyl, 1-propenyl, 1-butenyl optionally substituted by halogenor an alkynyl group.

By “1-alkynyl” is meant an acetylenically-unsaturated acylic group, suchas ethynyl, 1-propynyl, 1-butynyl, 1-pentynyl, 1,3-pentadiynyl, and thelike optionally substituted by an aryl or heteroaryl group, such asphenylethynyl, pyridine-ethynyl, pyrimidine-ethynyl, triazine-ethynyl,thiophene-ethynyl, thiazole-ethynyl and imidazole-ethynyl.

By “heteroaromatic” is meant a compound having at least one heterocyclicring having physical and chemical properties resembling compounds suchas an aromatic group of from 5 to 6 ring atoms and 2 to 12 carbon atomsin which one to three ring atoms is N, S or O, for example,2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 2-thiazoyl, triazinyl,2-imidazolyl, 2-oxazolyl, 2-pyridinyl (o-pyridinyl), 3-pyridinyl(m-pyridinyl), 4-pyridinyl (p-pyridinyl), 2-thienyl, 2-furanyl,2-pyrrolyl optionally substituted preferably on a ring C by oxygen,alkyl of 1-4 carbon atoms or halogen or on a ring N by alkyl of 1-4carbon atoms. Preferred substituents on the heteroaryl group are methyl,ethyl, trifluoromethyl, and bromo.

Preferred oligomers contain one or more S—R²-U or 5-R²—C bases.

In another embodiment of the invention, the oligomer comprises at leastone base of formula (1) or (2) wherein each X is independently O or S;and R² is selected from the group consisting of phenylethynyl, 2-, 3-,and 4-pyridine-ethynyl, 2-, 4- and 5-pyrimidine-ethynyl,triazine-ethynyl, 2-, 4- and 5-pyrimidinyl, 2-, 4- and 5-thiazolyl,1-methyl-2-imidazolyl, 2- and 4-imidazolyl, 2-, 4- and 5-oxazolyl,3-pyridinyl, 4-pyridinyl, 2-pyridinyl, 2- and 3-furanyl-ethynyl, 2- and3-thienyl-ethynyl, 2- and 4-imidazolyl-ethynyl, 2-, 4- and5-thiazoyl-ethynyl, 2-, 4- and 5-oxazolyl-ethynyl, 2- and3-pyrrolyl-ethynyl, 2- and 3-thienyl, 2- and 3-furanyl, 2- and3-pyrrolyl, propenyl (—CH═CH—CH₃), vinyl and —C≡C—Z where Z is hydrogen(H) or C₁₋₁₀ alkyl, haloalkyl (C₁₋₁₀ with 1 to 6 halogen atoms orheteroalkyl (C₁₋₁₀ with 1 to 3 heteroatoms selected from the groupconsisting of O, N and S); and

Pr is (H)₂ or a protecting group.

Examples of —C═C—Z include 1-propynyl (—C≡C—CH₃), 3-buten-1-ynyl(—C≡C—CH═CH₂), 3-methyl-1-butynyl (—C≡C—CH(CH₃)₂),3,3-dimethyl-1-butynyl (—C≡C—C(CH₃)₃), 1-butynyl (—C≡C—CH₂—CH₃),1,3-pentadiynyl (—C≡C—C≡C—CH₃) and ethynyl.

Preferred halogens are selected from the group consisting of fluorine,chlorine and bromine. Substitutions including bromovinyl can be includedin the oligomers.

Aspects of the invention include the use of nucleomonomers, two linkednucleomonomers (dimers), three linked nucleomonomers (trimers), fourlinked nucleomonomers (tetramers), five linked nucleomonomers(pentamers) or six linked nucleomonomers (hexamers) as intermediates inthe synthesis of the longer oligomers of the invention. These oligomersare valuable synthons of the invention that are useful in the synthesisof longer oligomers.

In other aspects, the invention is directed to duplexes or triplexesobtained by binding the foregoing oligomers to single-stranded or duplexnucleic acid targets.

Other useful intermediates in the synthesis of the oligomers of theinvention include an o-xyloso dimer having the structural formula (5),

wherein each Y is independently an oligomer or R¹; and each B isindependently a base provided that at least one B is a base of formula(1) or (2) wherein R² is as defined herein.

Also included are intermediates of the formula (6) shown in FIG. 1,wherein X is selected from the group consisting of O and S; and each Y,B, and R³ is independently selected and has the meaning defined herein.

The oligomers of the invention are also suitable for binding to DNAduplex target sequences via either CT or GT triple helix binding motif.

The novel oligomers of the present invention are useful in antisensetherapies wherein an oligomer hydridizes with a selected complementaryRNA sequence or triple helix therapies wherein an oligomer hydridizeswith a selected complementary DNA sequence.

The invention is also directed to an oligomer of the inventioncomprising a positive modification of least one base of formula (1) or(2) of the invention and a negative modification, each with respect tothe binding affinity of the oligomer to a complementary nucleic acidsequence. The positive modification counteracts the effect of thenegative modification to a degree that is more than additive withrespect to the binding affinity, thus a synergistic effect is observed.

An aspect of the invention is the inclusion of the invention bases inoligomers that are resistant to nuclease degradation relative to anoligodeoxynucleotide having no modifications. Nuclease resistantoligomers of the invention are advantageously used under conditionswhere nucleases are present. For certain applications, such asmodulation of gene expression by via an antisense mechanism, nucleasestability by oligomers of the invention is an important functionalaspect of the oligomer.

Other aspects of the invention are directed to pharmaceuticalcompositions, reagents and kits comprising the oligomers of theinvention, to methods of treating conditions, such as cancers andviruses or the like. Such conditions are associated with orcharacterized by particular nucleic acids such as DNA duplexes orsingle-stranded RNA or DNA.

An additional aspect of the invention includes methods of detecting thepresence, absence or amount of a particular single-stranded DNA or RNAor a particular target duplex in a biological (or other) sample usingthe oligomers of the invention, to detect selected nucleic acidsequences. Such sequences can be associated with the presence ofneoplastic growth, viruses or disease conditions. Reagents and kitscontaining oligomers of the invention represent an aspect of theinvention that permit facile use of the oligomers as reagents useful for(1) modulating gene expression in cells in vitro including cells grownin tissue culture, and

-   -   (2) detecting and/or quantitating target sequences.

It has been found that some of the oligomers of the invention haveenhanced binding properties with respect to complementarysingle-stranded and double-stranded nucleic acid sequences as comparedto unmodified oligomers not having the 5-substitution of the invention.Triple helix structures can be formed at physiological pH levels of 7.0and higher, where unmodified control oligomers were less efficient.Improved duplex formation is also noted.

A feature of the invention is that the oligomers of the invention can becomprised of a variety of different sequences and thereby used to targeta variety of different single-stranded or double-stranded targetsequences.

An advantage of the present invention is that the oligomers of theinvention are capable of forming triplexes under physiological pHconditions.

Another feature of oligomers containing 5-R² substituted uracil orcytosine base (1) or (2) of the invention compared to oligomerscontaining thymine or cytosine is that the lipophilic group (R²) canenhance cell permeation or uptake. The nucleomonomers containing thesebases are more lipophilic than uridine, cytidine or thymidine based onretention times on reverse phase HPLC.

Additional Nucleomonomer Modifications.

Oligomers that are comprised of nucleomonomers can also containmodifications in addition to the 5-modified pyrimidines of theinvention. A non-limiting exemplary list of such additionalmodifications includes oligomers where (i) one or more nucleomonomerresidues are modified at the 2′ position, (ii) one or more covalentcrosslinking moieties are incorporated, (iii) inverted polarity linkersare incorporated, (iv) substitute linkages are included, (v) other baseanalogs, such as 8-oxo-N⁶-methyladenine, are included and (vi)conjugates such as intercalating agents or polylysine that respectivelyenhance binding affinity to target nucleic acid sequences or thatenhance association of the oligomer with cells are included.

The ability of the 5-substitution of the bases (1) and (2) of theinvention to enhance affinity of the oligomer for single-stranded andduplex targets (positive modification) permits further modifications tothe oligomer in which they are contained. These further modificationsmay or may not diminish affinity, but also confer other usefulproperties such as stability to nuclease cleavage, ability to permeatecell membranes, and the like. Any decrease in binding affinity resultingfrom the further modifications (negative modification) is acceptablebecause of the enhanced affinity conferred by the 5-substituted bases(1) and (2). Thus, particularly preferred oligomers of the invention cancontain substitute linkages and/or modified sugars, as well as the5-substituted pyrimidine bases (1) and (2) of the invention.

The oligomers can also contain additional modifications in thenucleomonomers that contain these 5-modified pyrimidines or in othernucleomonomers that comprise the oligomer.

Also included are oligomers containing one or more substitute linkagessuch as sulfide or sulfone linkages (Benner, S. A., InternationalPublication No. WO 89/12060), sulfamate linkages (InternationalPublication No. WO 91/15500), carbamate linkages in morpholino-linkedoligomers (Stirchak, E. P. et al Nucleic Acids Res (1989) 17: 6129-6141)and related linkages in morpholino oligomers of the formula (7) shown inFIG. 1 wherein X² is CO, CS or SO₂; X³: is O, S, NH, NCH₃, CH₂, CF² orCHF; each Y is independently an oligomer or R¹ and each B isindependently chosen and has the previously defined meaning, providedthat at least one B is a base of formula (1) or (2).

Riboacetal and related linkages, amide linkages and 2′,5′ linkages aredescribed in commonly owned pending U.S. application Ser. No.07/806,710, filed Dec. 12, 1991, Ser. No. 07/899,736, filed Can 28,1992, Ser. No. 07/894,397 and filed 07/892,902, filed each citedreference is incorporated herein by reference.

Exemplary dimers containing riboacetal and related linkages of formulae(8-15) are shown in FIGS. 2 and 3 wherein for each structure,

-   -   R¹ and B are independently chosen and have the meanings defined        above;    -   R³ has the meaning as defined above;    -   X³ is independently selected from the group consisting of O, S,        NH, NCH₃, CH₂, CF₂ and CFH;    -   X⁴ is independently selected from the group consisting of O, S,        SO, SO₂, CH₂, CO, CF₂, CS, NH and NR⁴ wherein R⁴ is lower alkyl        (C₁₋₄; methyl, ethyl, propyl, isopropyl, butyl or isobutyl);    -   X⁵ is selected from the group consisting of O, CO, S, CH₂, CS,        NH and NR⁴;    -   X⁶ is selected from the group consisting of CH, N, CF, CCl, and        CR⁵ wherein R⁵ is methyl or lower alkyl (C₂₋₄) fluoromethyl,        difluoromethyl, trifluoromethyl or lower fluoroalkyl (C₂₋₄,        F₁₋₅);    -   X⁷ is selected from the group consisting of O, S, CH₂, CO, CF₂        and CS;    -   provided that at least one B is of the formula (1) or (2) as        defined above; and    -   further provided that no adjacent X⁴, X⁵ or X⁷ are O (i.e.,        —O—O—, a peroxide).

Compounds of the 5-member ring series are preferred embodiments foroligomers containing one or more riboacetal linkages (formula (8)),where X⁴ is O and X⁵, X⁷ are CH₂ and X⁶ is CH.

Also included are oligomers containing nucleomonomer residues linked viaamide bonds. Exemplary linkages have been described (Nielsen, P. E., etal, Science (1991) 254: 1497-1500; commonly owned copending U.S.application Ser. No. 07/889,736, filed Can 28, 1992, and Ser. No.07/894,397, filed Jun. 5, 1992, both incorporated herein by reference).

Oligomers

As used herein “oligomer” includes oligonucleotides, oligonucleosides,polydeoxyribo-nucleotides (containing 2′-deoxy-D-ribose or modifiedforms thereof), i.e., DNA, polyribonucleotides (containing D-ribose ormodified forms thereof), i.e., RNA, and any other type of polynucleotidewhich is an N-glycoside or C-glycoside of a purine or pyrimidine base,or modified purine or pyrimidine base. Oligomer as used herein is alsointended to include compounds where adjacent nucleomonomers are linkedvia amide linkages as previously described (Nielsen, P. E., et al,Science (1991) 254: 1497-1500). The enhanced competence of binding byoligomers containing the bases of the present invention is believed tobe primarily a function of the base alone. Because of this, elementsordinarily found in oligomers, such as the furanose ring and/or thephosphodiester linkage can be replaced with any suitable functionallyequivalent element. “Oligomer” is thus intended to include any structurethat serves as a scaffold or support for the bases wherein the scaffoldpermits binding to target nucleic acids in a sequence-dependent manner.Oligomers that are currently known can be defined into four groups thatcan be characterized as having (i) phosphodiester and phosphodiesteranalog (phosphorothioate, methylphosphonate, etc) linkages, (ii)substitute linkages that contain a non-phosphorous isostere (formacetal,riboacetal, carbamate, etc), (iii) morpholino residues, carbocyclicresidues or other furanose sugars, such as arabinose, or a hexose inplace of ribose or deoxyribose and (iv) nucleomonomers linked via amidebonds or acyclic nucleomonomers linked via any suitable substitutelinkage.

The oligomers of the invention can be formed using invention andconventional nucleomonomers and synthesized using standard solid phase(or solution phase) oligomer synthesis techniques, which are nowcommercially available. In general, the invention oligomers can besynthesized by a method comprising the steps of: synthesizing anucleomonomer or oligomer synthon having a protecting group and a baseand a coupling group capable of coupling to a nucleomonomer or oligomer;coupling the nucleomonomer or oligomer synthon to an acceptornucleomonomer or an acceptor oligomer; removing the protecting group;and repeating the cycle as needed until the desired oligomer issynthesized.

The oligomers of the present invention can be of any length includingthose of greater than 40, 50 or 100 nucleomonomers. In general,preferred oligomers contain 2-30 nucleomonomers. Lengths of greater thanor equal to about 8 to 20 nucleomonomers are useful for therapeutic ordiagnostic applications. Short oligomers containing 2, 3, 4 or 5nucleomonomers are specifically included in the present invention andare useful as synthons.

Oligomers having a randomized sequence and containing about 6 or 7nucleomonomers are useful for primers that are used in cloning oramplification protocols that use random sequence primers, provided thatthe oligomer contains residues that can serve as a primer forpolymerases or reverse transcriptases.

Oligomers can contain conventional phosphodiester linkages or cancontain substitute linkages such as phosphoramidate linkages. Thesesubstitute linkages include, but are not limited to, embodiments whereina moiety of the formula —O—P(O)(S)—O-(“phosphorothioate”),—O—P(S)(S)—O-(“phosphorodithioate”), —O—P(O)(NR′₂)—X—, —O—P(O)(R′)—O—,—O—P(S)(R′)—O— (“thionoalkylphosphonate”), —P(O)(OR⁶)—X—, —O—C(O)—X—, or—O—C(O)(NR₁₂)—X—, wherein R′ is H (or a salt) or alkyl (1-12C) and R⁶ isalkyl (1-9C) and the linkage is joined to adjacent nucleomonomersthrough an —O— or —S— bonded to a carbon of the nucleomonomer.Phosphorothioate and phosphodiester linkages are shown in FIG. 12.Particularly, preferred substitute linkages for use in the oligomers ofthe present invention include phosphodiester, phosphorothioate,methylphosphonate and thionomethylphosphonate linkages. Phosphorothioateand methylphosphonate linkages confer added stability to the oligomer inphysiological environments. While not all such linkages in the sameoligomer need be identical, particularly preferred oligomers of theinvention contain uniformly phosphorothioate linkages or uniformlymethylphosphonate linkages.

Pharmaceutically Acceptable Salts

Any pharmaceutically acceptable salt can be used and such salt formingmaterials are well known in the art.

Pharmaceutically acceptable salts are preferably metal or ammonium saltsof said oligomers of the invention and include alkali or alkaline earthmetal salts, e.g., the sodium, potassium, magnesium or calcium salt; oradvantageously easily crystallizing ammonium salts derived from ammoniaor organic amines, such as mono-, di- or tri-lower (alkyl, cycloalkyl orhydroxyalkyl)-amides, lower alkylenediamines or lower (hydroxyalkyl orarylalkyl)-alkylammonium bases, e.g. methylamine, diethylamine,triethylamin, dicyclohexylamine, triethanolamine, ethylenediamine,tris-(hydroxymethyl)-aminomethane or benzyl-trimethylammonium hydroxide.The oligomers of the invention form acid addition salts, which arepreferably such of therapeutically acceptable inorganic or organicacids, such as strong mineral acids, for example hydrohalic, e.g.,hydrochloric or hydrobromic acid; sulfuric, phosphoric; aliphatic oraromatic carboxylic or sulfonic acids, e.g., formic, acetic, propionic,succinic, glycollic, lactic, malic, tartaric, gluconic, citric,ascorbic, maleic, fumaric, hydroxymaleic, pyruvic, phenylacetic,benzoic, 4-aminobenzoic, anthranilic, 4-hydroxybenzoic, salicylic,4-aminosalicylic, methanesulfonic, ethanesulfonic,hydroxyethanesulfonic, benzenesulfonic, sulfanilic or cyclohexylsulfamicacid and the like.

Blocking Groups

As used herein, “blocking group” refers to a substituent other than Hthat is conventionally coupled to oligomers or nucleomonomers, either asa protecting group, a coupling group for synthesis, PO₃ ⁻², or otherconventional conjugate such as a solid support, label, antibody,monoclonal antibody or fragment thereof and the like. As used herein,“blocking group” is not intended to be construed solely as a protectinggroup, according to slang terminology, but is meant also to include, forexample, coupling groups such as a H-phosphonate or a phosphoramidite.

By “protecting group” is meant is any group capable of protecting theO-atom or N-atom to which it is attached from participating in areaction or bonding. Such protecting groups for N-atoms on a base moietyin a nucleomonomer and their introduction are conventionally known inthe art. Non-limiting examples of suitable protecting groups includediisobutylformamidine, benzoyl and the like. Suitable “protectinggroups” for O-atoms are, for example, DMT, MMT, or FMOC.

Suitable coupling groups are, for example, H-phosphonate, amethylphosphonamidite, or a phosphoramidite. Phosphoramidites that canbe used include β-cyanoethylphosphoramidites (preferred).Methylphosphonamidites, alkylphosphonamidites (includingethylphosphonamidites and propylphosphonamidites) can also be used.Exemplary phosphoramidites are shown in FIGS. 10-1 and 10-2.

Suitable protecting groups are DMT (dimethoxy trityl), MMT(monomethoxytrityl) or FMOC at the 5′ terminus and/or hydrogenphosphonate, methyl phosphoramidite, methyl phosphonamidite,β-cyanoethylphosphoramidite at the 3′-terminus.

Protecting Groups

Protecting groups such as diisobutylformamidine, benzoyl, isobutyryl,FMOC, dialkylformamidine, dialkylacetamidine or other groups known inthe art can be used to protect the exocyclic nitrogen of the cytosineheterocycle. Alternatively, cytidine precursors can be directlyincorporated into oligomers without a protecting group at the exocyclicnitrogen using described methods (Gryaznov, S. M. et al, J Amer Chem Soc(1991) 113: 5876-5877; Gryaznov, S. M., et al, Nucl Acids Res (1992) 20:1879-1882; Kung, P.-P., et al, Tetrahedron Letters (1992) 40:5869-5872). Synthesis of oligomers having bases (1) or (2) containing anR² as ethynyl heteroaryl substituents is preferably accomplished using9-fluorenylmethoxycarbonyl (FMOC) for protection of the 5′-hydroxylposition as described (Lehman, C., et al, Nucl Acids Res (1989) 17:2379-2390).

Preferred protecting groups are DMT (dimethoxy trityl), MMT(monomethoxytrityl) or FMOC at the 5′ terminus and/or hydrogenphosphonate, methyl phosphoramidite, methyl phosphonamidite,β-cyanoethylphosphoramidite at the 3′-terminus. However, it is intendedthat the position of the blocking groups can be reversed as needed(e.g., a phosphoramidite at the 5′-position and DMT at the 3′-position).In general, the nucleomonomers and oligomers of the invention can bederivatized to such “blocking groups” as indicated in the relevantformulas by methods known in the art.

Coupling Groups

Suitable coupling groups are, for example, H-phosphonate, amethylphosphonamidite, or a phosphoramidite. Phosphoramidites that canbe used include β-cyanoethylphosphoramidites (preferred).Methylphosphonamidites, alkylphosphonamidites (includingethylphosphonamidites and propylphosphonamidites) can also be used.Exemplary phosphoramidites are shown in FIGS. 10-1 and 10-2. Suitable“coupling groups” at the 3′, 2′ or 5′ position for oligomer synthesisvia phosphoramidite triester chemistry, referred to herein as “amidite”chemistry, include N,N-diisopropylamino-β-cyanoethoxyphosphine,N,N-diisopropylamino-methoxyphosphine,N,N-diethylamino-β-cyanoethoxyphosphine,(N-morpholino)-β-cyanoethoxyphosphine, and(N-morpholino)-methoxyphosphine (Moore, M. F. et al, J Org Chem (1985)50: 2019-2025; Uznanski, A. W., et al, Tet Lett (1987) 28: 3401-3404;Bjergarde, K., et al, Nucl Acids Res (1991)21: 5843-5850; Dahl, O.Sulfur Reports (1991) 11: 167-192). Related coupling groups such asN,N-diisopropylamino-methyl-phosphine orN,N-diethylamino-methyl-phosphine can also be used to preparemethylphosphonates (FIG. 10-4). Methylphosphonate oligomers can beconveniently synthesized using coupling groups such asN,N-diisopropylamino-methylphosphonamidite, andN,N-diethylamino-methylphosphonamidite. Synthesis of nucleomonomeramidites of the invention can be accomplished by conventional methods(for example, Gryaznov, S. M., et al, Nucl Acids Res (1992) 20:1879-1882; Vinayak, R., et al, Nucl Acids Res (1992) 20: 1265-1269;Sinha, N. D., et al, Nucl Acids Res (1984) 12: 4539-4557; and otherreferences cited herein). Suitable coupling groups at the 3′, 2′ (or 5′)position for oligomer synthesis via phosphate triester chemistry,referred to herein as “triester” chemistry, include 2-chlorophenylphosphate, 4-chlorophenyl phosphate, 2,4-dichlorophenyl phosphate and2,4,-dibromophenyl phosphate nucleotide diester derivatives or, forsynthesis of phosphorothioate linkages, the thiono derivatives thereof(Marugg, J. E., et al, Nucl Acids Res (1984) 12: 9095-9110; Kemal, O.,et al, J Chem Soc Chem Commun (1983) 591-593; Kamer, P. C. J., et al,Tet Lett (1989) 30: 6757-6760). Structures of these coupling groups areshown in FIG. 10 where X is O or S and Z¹ is H or a suitablebenzotriazole.

Oligomers or the segments thereof are conventionally synthesized. Thesynthetic methods known in the art and described herein can be used tosynthesize oligomers containing bases of the invention, as well as otherbases known in the art, using appropriately protected nucleomonomers(see FIG. 12). Methods for the synthesis of oligomers are found, forexample, in Froehler, B., et al., Nucleic Acids Res (1986) 14:5399-5467; Nucleic Acids Res (1988) 16: 4831-4839; Nucleosides andNucleotides (1987) δ: 287-291; Froehler, B., Tetrahedron Letters (1986)27: 5575-5578; Caruthers, M. H. in Oligodeoxynucleotides-AntisenseInhibitions of Gene Expression (1989), J. S. Cohen, editor, CRC Press,Boca Raton, p 7-24; Reese, C. B. et al, Tetrahedron Letters (1985) 26:2245-2248. Synthesis of the methylphosphonate linked oligomers viamethyl phosphonamidite chemistry has also been described (Agrawal, S. etal., Tetrahedron Letters (1987) 28: 3539-3542; Klem, R. E., et al,International Publication Number WO 92/07864).

Conjugates

Also included are “conjugates” of oligomers. “Conjugates” of theoligomers include those conventionally recognized in the art. Forinstance, the oligomers can be covalently linked to various moietiessuch as, intercalators, and substances which interact specifically withthe minor groove of the DNA double helix. Other chosen conjugatemoieties, can be labels such as radioactive, fluorescent, enzyme, ormoieties which facilitate cell association using cleavage linkers andthe like. Suitable radiolabels include ³²P, ³⁵S, ³H and ¹⁴C; andsuitable fluorescent labels include fluorescein, resorufin, rhodamine,BODIPY (Molecular Probes) and texas red; suitable enzymes includealkaline phosphatase and horseradish peroxidase. Other compounds whichcan be used as covalently linked moieties include biotin, antibodies orantibody fragments, transferrin and the HIV Tat protein can alsoconveniently be linked to the oligomers of the invention.

These additional moieties can be derivatized through any convenientlinkage. For example, intercalators, such as acridine or psoralen can belinked to the oligomers of the invention through any available —OH or—SR, e.g., at the terminal 5′-position of the oligomer, the 2′-positionsof RNA, or an OH, NH₂, COOH or SH incorporated into the 5position ofpyrimidines. A derivatized form which contains, for example, —CH₂CH₂NH₂,—CH₂CH₂CH₂OH or —CH₂CH₂CH₂SH in the 5position is preferred. Conjugatesincluding polylysine or lysine can be synthesized as described and canfurther enhance the binding affinity of an oligomer to its targetnucleic acid sequence (Lemaitre, M. et al., Proc Natl Acad Sci (1987)84:648-652; Lemaitre, M. et al., Nucleosides and Nucleotides (1987) δ:311-315).

A wide variety of substituents can be attached, including those boundthrough linkages or substitute linkages. The —OH moieties in theoligomers can be replaced by phosphate groups, protected by standardprotecting groups, or coupling groups to prepare additional linkages toother nucleomonomers, or can be bound to the conjugated substituent. The5′terminal OH can be phosphorylated; the 2′-OH or OH substituents at the3′-terminus can also be phosphorylated. The hydroxyls can also bederivatized to standard protecting groups.

Oligomers of the invention can be covalently derivatized to moietiesthat facilitate cell association using cleavable linkers. Linkers usedfor such conjugates can include disulfide linkages that are reducedafter the oligomer-transport agent conjugate has entered a cell.Appropriate molecular linkers include for example,—Y¹—X⁸CH₂CHR⁷—SS—CHR⁷CH₂X⁸—Y¹— wherein each Y¹ is independently alkylene(C₁₋₆; including methylene, ethylene and propylene), or CO, each X⁸ isindependently O, S(O)(O), S(O), NR⁷, CH₂, C(R⁷)₂ or CO; R⁷ wherein eachR⁷ is independently H, alkyl (C₁₋₆; including methyl, ethyl and propyl),or aryl and which linkers have been previously described (Wo 91/14696).Disulfide-containing linkers of this type have a controllable t_(1/2) invivo, facilitating its use as a prodrug/transport component. Suchlinkers are stable under extracellular conditions relative tointracellular conditions due to the redox potential of the disulfidelinkage.

Suitable conjugates also include solid supports for oligomer synthesisand to facilitate detection of nucleic acid sequences. Solid supportsincluded, but are not limited to, silica gel, controlled pore glass,polystyrene, and magnetic glass beads.

Sugar Modifications.

Derivatives can be made by substitution on the sugars. Among the mostpreferred derivatives of the oligomers of the invention are the2′-O-allyl derivatives. The presence of the 2′-O-allyl group appears toenhance permeation ability and stability to nuclease degradation, butdoes not appear to diminish the affinity of the oligomer for singlechain or duplex targets.

Furthermore, as the α anomer binds to duplex DNA or single-stranded RNAin a manner similar to that for the β anomers but with a reversedpolarity, oligomers can contain nucleomonomers having this epimer or adomain thereof (Praseuth, D., et al., Proc Natl Acad Sci (USA) (1988)85: 1349-1353; Sun, J. S. et al, Proc Natl Acad Sci (1991) 88:6023-6027; Debart, F., et al, Nucl Acids Res (1992) 20: 1193-1200).α-Anomeric oligomers containing the 5-R² substituted pyrimidinesdescribed herein represent a class of modified oligomers included in thepresent invention.

Substitute Linkages

The oligomers of the invention can also contain one or more “substitutelinkages” as is generally understood in the art. These “substitutelinkages” include phosphorothioate, methylphosphonate,thionomethylphosphonate, phosphorodithioate, riboacetal, 2′, 5′linkages, alkylphosphonates, morpholino carbamate, morpholino sulfamate,morpholino sulfamide, boranophosphate (—O—P(OCH₃) (BH₃)—O—), siloxane(—O—Si(X⁴)(X⁴)—O—; X⁴ is alkyl or phenyl) and phosphoramidate(methoxyethylamine and the like), and are synthesized as described inthe generally available literature and in references cited herein (Sood,A., et al, J Am Chem Soc (1990) 112: 9000-9001; WO 91/08213; WO90/15065; WO 91/15500; Stirchak, E. P. et al Nucleic Acid Res (1989) 17:6129-6141; U.S. Pat. No. 5,034,506; U.S. Pat. No. 5,142,047; Hewitt, J.M. et al, Nucleosides and Nucleotides (1992) 11: 1661-1666). Substitutelinkages that can be used in the oligomers disclosed herein also includethe sulfonamide (—O—SO₂—NH—), sulfide (—CH₂—S—CH₂—), sulfonate(—O—SO₂—CH₂—), carbamate (—O—C(O)—NH—, —NH—C(O)—O—), dimethylhydrazino(—CH₂—NCH₃—NCH₃—), sulfamate (—O—S(O)(0)—N—; —N—S(O)(O)—N—),3′-thioformacetal (—S—CH₂—O—), formacetal (—O—CH₂—O—), 3′-amine(—NH—CH₂—CH₂—), N-methylhydroxylamine (—CH₂—NCH₃—O—) and 2′5′ linkages(such as 2′,5′ carbamate (2′ —N(H)—C(O)—O— 5′), 5′,2′ carbamate (2′-O—C(O)—N(H)— 5′), 5′,2′ methylcarbamate (2′-O—C(O)—N(CH₃)-5′) and 5′,2′thioformacetal (2′-O—CH₂—S-5′). 2′,5′ linkages are disclosed in pendingU.S. application Ser. No. 07/892,902, filed Jun. 1, 1992, incorporatedherein by reference). Riboacetal linkages are disclosed and claimed incommonly owned pending U.S. patent application Ser. No. 690,786, filedApr. 24, 1991, Ser. No. 763,130, filed Sep. 20, 1991, and Ser. No.806,710 filed Dec. 12, 1991, incorporated herein by reference. Exceptwhere specifically indicated, the substitute linkages, such as aformacetal linkage, —O—CH₂—O—, are linked to either the 3′ or 2′ carbonof a nucleomonomer on the left side and to the 5′ carbon of anucleomonomer on the right side. A formacetal linked (3′,5′) dimer isshown in FIG. 1, formula (6). Thus a formacetal linkage can be indicatedas 3′ —O—CH₂—O— 5′ or 2′ —O—CH₂—O— 5′. The designations of a 3′, 2′ or5′ carbon can be modified accordingly when a structure other thanribose, deoxyribose or arabinose is linked to an adjacent nucleomonomer.Such structures include a hexose, morpholino ring, carbocyclic ring(e.g. cyclopentane) and the like.

The use of carbamate, carbonate, sulfide, sulfoxide, sulfone,N-methylhydroxylamine and dimethylhydrazino linkages in synthons oroligomers has been described (Vaseur, J-J. et al, J Amer Chem Soc (1992)114: 4006-4007; WO 89/12060; Musicki, B. et al, J Org Chem (1990) δ:4231-4233; Reynolds, R. C., et al J Org Chem (1992) 57: 2983-2985;Mertes, N. P., et al, J Med Chem (1969) 12: 154-157; Mungall, W. S., etal, J Org Chem (1977) 42: 703-706; Stirchak, E. P., et al, J Org Chem(1987) δ: 4202-4206; Wang, H., et al, Tet Lett (1991) 50: 7385-7388;International Application No. PCT US91/03680). Substitute linkage(s) canbe utilized in the oligomers for a number of purposes such as to furtherfacilitate binding with complementary target nucleic acid sequencesand/or to increase the stability of the oligomers toward nucleases.

By “positive modification” is meant use of any modification of formula(1) or (2) of the invention which results in increased binding affinity.

By “negative modification” is meant an additional nucleomonomermodification of an oligomer comprising a base of formula (1) or (2)which results in a decrease in binding affinity or use of a substitutelinkage which may result in a decrease in binding affinity.

For example, a negative substitute linkage modification is at least oneselected from phosphorothioate, methylphosphonate,thionomethylphosphonate, phosphoroamidate and triester for aphosphodiester linkage.

Nucleosides

The term “nucleoside” will include ribonucleosides,deoxyribonucleosides, or to any other nucleoside which is an N-glycosideor C-glycoside of a purine or pyrimidine base, or modified purine orpyrimidine base. The stereochemistry of the sugar carbons can be otherthan that of D-ribose in one or more residues. The pentose moiety can bereplaced by a hexose and incorporated into oligomers as described(Augustyns, K., et al Nucl Acids Res (1992) 18: 4711-4716). Alsoincluded are analogs where the ribose or deoxyribose moiety is replacedby an alternate structure such as a hexose or such as the 6-membermorpholino ring described in U.S. Pat. No. 5,034,506. Nucleosides asdefined herein also includes a purine or pyrimidine base linked to anamino acid or amino acid analog having a free carboxyl group and a freeamino group or protected forms thereof. Exemplary nucleosides have beendescribed (Nielsen, P. E. ibid; commonly owned copending U.S.application Ser. No. 07/889,736, filed Can 28, 1992, and Ser. No.07/894,397, filed Jun. 5, 1992, both applications incorporated herein byreference in their entirety).

“Nucleosides” also include those moieties which contain modifications ofthe sugar, for example, wherein one or more of the hydroxyl groups arereplaced with halogen, aliphatic groups, or functionalized as ethers,amines, and the like. Such structures include a hexose, morpholino ring,carbocyclic ring (e.g. cyclopentane) and the like.

Base

“Base” as used herein includes those moieties which contain not only theknown purine and pyrimidine bases and the invention bases, but alsoheterocyclic bases which have been modified and tautomers thereof. Suchmodifications include alkylated purines or pyrimidines, acylated purinesor pyrimidines, or other heterocycles. Such “analogous purines” and“analogous pyrimidines” or purine or pyrimidine analogs are thosegenerally known in the art, some of which are used as chemotherapeuticagents. An exemplary, but not exhaustive, list includesN⁴,N⁴-ethanocytosine, 7-deazaxanthosine, 7-deazaguanosine,8-oxo-N⁶-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N⁶-isopentenyl-adenine, 1-methyladenine,1-methylpseudouracil, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N⁶-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy aminomethyl-2-thiouracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, pseudouracil, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, 2-thiocytosine, and2,6-diaminopurine. In addition to these base analogs, pyrimidine analogsincluding 6-azacytosine, 6-azathymidine and 5-trifluoromethyluracildescribed in Cook, D. P., et al, International Publication No. WO92/02258 (incorporated herein by reference) can be convenientlyincorporated into the invention oligomers.

Bases of formula (1) or (2) containing sulfur at the 2 and/or 4 positioncan be incorporated into oligomers and derivatized with alkynyl as R²essentially as described above. In corporation of 4-thiouridine and2-thiothymidine into oligomers has been described (Nikiforov, T. T., etal, Tet Lett (1992) 33: 2379-2382; Clivio, P., et al Tet Lett (1992) 33:65-68; Nikiforov, T. T., et al, Tet Lett (1991) 32: 2505-2508; Xu,Y.-Z., et al Tet Lett (1991) 32: 2817-2820; Clivio, P., et al Tet Lett(1992) 33: 69-72; Connolly, B. A., et al., Nucl. Acids Res. (1989) 17:4957-4974).

Preferred bases are of the formula (1) and (2) but also include adenine,guanine, thymine, uracil, cytosine, 5-methylcytosine,8-oxo-N⁶-methyladenine, pseudoisocytosine, and 7-deazaxanthosine.Synthesis and use of oligomers that bind to duplex DNA sequences via GTbinding motif containing 7-deazaxanthosine is described in commonlyowned pending U.S. application Ser. No. 07/787,920, filed Nov. 7, 1991,incorporated herein by reference.

Synthesis

Oligomers or the segments thereof are conventionally synthesized. Thesynthetic methods known in the art and described herein can be used tosynthesize oligomers containing bases of the invention, as well as otherbases known in the art, using appropriately protected nucleomonomers(see FIG. 12). Methods for the synthesis of oligomers are found, forexample, in Froehler, B., et al., Nucleic Acids Res (1986) 14:5399-5467; Nucleic Acids Res (1988) 16: 4831-4839; Nucleosides andNucleotides (1987) δ: 287-291; Froehler, B., Tetrahedron Letters (1986)27: 5575-5578; Caruthers, M. H. in Oligodeoxynucleotides-AntisenseInhibitions of Gene Expression (1989), J. S. Cohen, editor, CRC Press,Boca Raton, p 7-24; Reese, C. B. et al, Tetrahedron Letters (1985) 26:2245-2248. Synthesis of the methylphosphonate linked oligomers viamethyl phosphonamidite chemistry has also been described (Agrawal, S. etal., Tetrahedron Letters (1987) 28: 3539-3542; Klem, R. E., et al,International Publication Number WO 92/07864).

Oligomers of the invention containing bases of formula (1) or (2) andone or more substitute linkages can be synthesized by one or more offour general methods according to the reaction conditions required forsynthesis of a given substitute linkage. In the first method (#1),nucleomonomers containing bases of formula (1) or (2) are directlyincorporated into oligomers or a convenient fragment thereof usingstandard synthesis conditions and reagents. Exemplary schemes are shownin FIGS. 5 and 12 and exemplary linkages that can be made by method #1include phosphodiester, phosphorothioate, phosphoroamidate,methylphosphonate, phosphorodithioate, carbonate, morpholino carbamateand sulfonate.

Method #2 involves synthesis of short synthons (dimers, trimers, etc)starting with an appropriate precursor such as a 5-bromo or 5-iodoprecusor (as described below) which is subsequently converted to the C-5substituent of formula (1) or (2) and a synthon suitable forincorporation into oligomers. This approach is exemplified in FIGS. 7, 8and 11 and is suitable for synthesis of linkages includingN-methylhydroxylamine, dimethylhydrazo, sulfamate, carbamate, sulfonate,sulfonamide, formacetal thioformacetal and carbonate.

Synthesis method #3 starts with uridine or cytidine (unprotected orN-protected) nucleomonomers which is subsequently iodinated.Introduction of the R² group at the 5-position is accomplished withinthe synthetic route to the desired dimer or trimer synthon. Method #3 isexemplified in FIG. 9 and is suitable for synthesis of linkagesincluding N-methylhydroxylamine, dimethylhydrazino, sulfamate,formacetal, thioformacetal, riboacetal, sulfonate, sulfonamide,carbamate, carbonate and boranophosphate linkages.

Method #4 starts with either (1) uracil or cytosine base containing R₂,followed by conversion to a nucleomonomer suitable for incorporationinto oligomers (e.g. amide linkages) as exemplified in FIG. 12 or (2) asuitable precusor such as 5-iodocytosine, 5-iodouracil, cytosine oruracil which is glycosylated or alkylated followed by conversion of thenucleomonomer to a derivative containing R² and converted to the desiredsynthon (e.g. linkages such as sulfide, sulfoxide or sulfonate).

In general, reaction conditions that are needed to synthesize aparticular dimer, trimer or larger synthon and may not be compatiblewith an R² alkynyl 2′-deoxyuridine or 2′-deoxycytidine are (1)electrophilic addition reactions, or conditions that could promoteelectrophilic addition to C—C multiple bonds (e.g. HCl, HF, BF₃, Cl₂ orBr₂); (2) conditions that could promote reduction of C—C multiple bonds(e.g., hydrogenation via H₂/Pd/C or hydrides such as B₂R₆, BH₃.complexor as a class, tin hydrides or aluminum hydrides); (3) conditions thatpromote free radical reactions (e.g., Cl₂hv, peroxides or AIBN); and (4)reaction conditions that promote oxidation of C—C multiple bonds (e.g.KMnO₄, OsO₄, alkyl carboxylic peracids). Synthetic schemes involvingthese reaction conditions may prevent the use of Method #1.

In general, reaction conditions that are required to synthesize certainoligomers that may not compatible with 5-iodo-2′-deoxyuridine or5-iodo-2′-deoxycytidine, or the like, are (1) conditions that promotereduction of aryl iodides (e.g., H₂ or hydrides), (2) alkylation andarylation reactions mediated by organometallic reagents or (3) reactionsthat promote free radical reactions (e.g., Cl₂hv, peroxides or AIBN).Synthetic schemes involving these reactions may prevent use of Method#2.

Method #3 starts with 2′-deoxyuridine or 2′-deoxycytidine and thenucleomonomer is subsequently converted to the 5-iodo (or 5-bromo or5-triflate) derivated (Robins, M. J. et al, Can. J. Chem (1982) 60:554-557; Chang, P. K. et al, J Med Chem (1963) 6: 428-430; Crisp, G. T.,et al., Tet Lett (1990) 31: 1347-1350; and Torrence, P. F., et al., J.Med. Chem (1979) 22: 316-319) at the desired step, followed byconversion to a R² substituent at desired step. In some schemes it isadvantageous to convert 2′-deoxyuridine to 5-R²-2′-deoxycytidine asneeded by previously desired methods (Divakar, K. J., et al, J. Chem.Soc. Perkin Trans I (1982) p 1171-1176). Where one of these reactions orconditions are used for synthesis of a given oligomer or fragmentthereof, a nucleomonomer such as 2′-deoxyuridine can be utilizedfollowed by conversion to the R² derivative and the cytidine derivativesthereof.

Additional exemplary linkages that can be synthesized by these generalmethods are summarized in Table A below.

TABLE A Linkage Structure* Method Reference** 2′ —S—CH₂— 5′ 1-4 1 3′—S—CH₂— 5′ 1-4 2 2′ —S(O)—CH₂— 5′ 1-4 1 3′ —S(O)—CH₂— 5′ 1-4 1 2′—S(O)(O)—CH₂— 5′ 1-4 1 3′ —S(O)(O)—CH₂— 5′ 1-4 1 2′ —CH₂—S— 5′ 3, 4 1 3′—CH₂—S— 5′ 3, 4 2 2′ —CH₂—S(O)— 5′ 3, 4 1 3′ —CH₂—S(O)— 5′ 3, 4 2 2′—CH₂—S(O)(O)— 5′ 3, 4 1 3′ —CH₂—S(O)(O)— 5′ 3, 4 2 2′ —CH₂—CH₂—O— 5′ 3,4 1 3′ —CH₂—CH₂—O— 5′ 3, 4 2 2′ —N(C(O)(OR^(A)))—CH₂—CH₂— 5′ 3, 4 1 3′—N(C(O)(OR^(A)))—CH₂—CH₂— 5′ 3, 4 2 2′ —S—CH₂—CH₂— 5′ 3, 4 1 3′—S—CH₂—CH₂— 5′ 3, 4 2 2′ —NH—C(O)—O— 5′ 3, 4 1 2′ —O—CH₂—S— 5′ 2-4 1 2′—O—C(O)—N(R^(B))— 5′ 2-4 1 5′ morpholino N—CH₂— 5′ 1-4 2

2-4 3

2-4 3

2-4 3

2-4 3

2-4 3

2-4 3 *R^(A) = C₁₋₆ alkyl, e.g. CH₂CH₃ or (CH₂)₅CH₃; R^(B) = H or C₁₋₆alkyl, e.g. CH₃; X = O or S; R^(C) = C₁₋₆ alkyl, CN or C₁₋₆ haloalkyl,e.g. CF₃; the linkages indicate covalent attachment of the indicatedatom with either a 2′, 3′ or 5′ carbon of ribose or deoxyribose. **1 -Synthesis is accomplished essentially as described in PCT/US91/06855 forequivalent 3′, 5′ linkages. 2 - International Application NumberPCT/US91/06855. 3 - International Application Number PCT/US90/06110;linkages having a structure such as C((CH₂)₂(CH₂)₂O) are cyclic ketals.

In addition to the substitute linkages given in Table A, FIG. 17 shows aseries of repeating nucleomonomer units (17-1) and exemplary amidelinked oligomers (17-2, 17-3) containing selected repeating units thatcan contain the base analogs of the invention. In FIG. 17-1, X⁹ is S, O,SO, SO₂, CH₂, CHF, CF₂ or NR¹⁰ and R¹⁰ is (independently) H, F, OH,OCH₃, CH₃, or CH-lower alkyl provided that adjacent X⁹ are not both O.In FIGS. 17-2 and 17-3, each Y is independently selected and has themeaning described above (e.g. Y is H, an oligomer, a blocking group suchas FMOC, tBOC, OH, DMT, MMT or an coupling group suitable for oligomersynthesis). Nucleomonomers required to synthesize oligomers containingsuch linkages are synthesized by method #4.

Oligomers of the invention can be synthesized by any suitable chemistryincluding amidite, triester or hydrogen phosphonate coupling methods andconditions. The oligomers are preferably synthesized from appropriatestarting synthons such as nucleomonomers of formula (3) or (4) whereinR¹ at the 5′-position is DMT, MMT, FMOC (9-fluorenylmethoxycarbonyl),PACO (phenoxyacetyl), a silyl ether such as TBDMS (t-butyldiphenylsilyl)or TMS (trimethylsilyl) and R¹ at the 3′-position is an ester,H-phosphonate, an amidite such as β-cyanoethylphosphoramidite, a silylether such as TBDMS or TMS or t-butyldiphenyl. Alternatively,appropriate uridine or cytidine precursors such as blocked5-iodo-2′-deoxyuridine, 5-iodo-2′-O-alkyluridine,5-bromo-2′-deoxyuridine, 5-trifluoromethanesulfonate-2′-deoxyuridine,5-bromo-2′-O-alkyluridine or blocked and protected5-iodo-2′-deoxycytidine, 5-bromo-2′-deoxycytidine,5-trifluoromethanesulfonate-2′-deoxycytidine, 5-iodo-2′-O-alkylcytidine,5-bromo-2′-O-alkylcytidine can be conveniently incorporated into shortoligomers such as dimer, trimer, tetramer, pentamer or longer synthonsthat are subsequently derivatized to yield R² at the 5-position and thenincorporated into suitable synthons and longer oligomers.

Synthesis of oligomers containing about 4 or more nucleomonomer residuesis preferably accomplished using synthons such as monomers, dimers ortrimers that carry a coupling group suitable for use with amidite,H-phosphonate or triester chemistries. The synthon can be used to linkthe oligomer via a phosphodiester or phosphorous-containing substitutelinkage (phosphorothioate, methylphosphonate, thionomethylphosphonate,phosphoramidate and the like).

Synthesis of other nonphosphorous-containing substituted linkages can beaccomplished using appropriate precursors as described herein (FIGS.7-10) and are known in the art.

In addition to employing these very convenient and now most commonlyused, solid phase synthesis techniques, oligomers can also besynthesized using solution phase methods such as triester synthesis.These methods are workable, but in general, less efficient for oligomersof any substantial length.

Intermediates or Starting Materials

In other aspects, the invention is directed to intermediates in thesynthesis of the oligomers of the invention, including nucleomonomeranalogs of formula (3) or (4):

wherein each R¹ is independently H or a blocking group;

-   -   R² and X are as defined above;    -   R³ is selected from the group consisting of H, OH, F, NH₂, OR or        SR, wherein OR is O-allyl or SR is S-allyl or O or S alkyl        (C₁₋₃), wherein alkyl including methyl, ethyl and propyl); and    -   Pr is (H)₂ or a protecting group;    -   provided that if X is O, R³ is H or OH, and both R¹ are H, then        R² is 1,3-pentadiynyl, 2-, 3- and 4-pyridine-ethynyl, 2-, 4- and        5-pyrimidine-ethynyl, triazine-ethynyl, arylethynyl, 2-, 4- and        5-pyrimidinyl, 2- and 4-imidazolyl, 2- and 3-pyrrolyl-ethynyl,        2- and 3-furanyl-ethynyl, 2- and 3-thienyl-ethynyl, 2- and        4-imidazolyl-ethynyl, 2-, 4- and 5-thiazoyl-ethynyl or 2-, 4-        and 5-oxazolyl-ethynyl, 4-oxazolyl, 4-thiazoyl, 3-pyrroyl,        3-furanyl, 3-thienyl and    -   further provided that for formula 3, when X is O, and R¹ and R³        are H, then R² is not vinyl, 1-butenyl, 1-pentenyl, 1-hexenyl,        1-heptenyl, 1-octenyl, 1-propynyl, 1-butynyl, 1-hexynyl,        1-heptynyl or 1-octynyl.

Suitable protecting groups (Pr) include diisopropylformanidine,di-n-butylformanidine, diisobutylformamidine and benzoyl and suitable R¹groups including DMT, MMT, FMOC, a phosphoramidite such asβ-cyanoethylphosphoramidite, hydrogen-phosphonate andmethylphosphonamidite.

Preferred protected nucleomonomers are nucleomonomers of formula (3) and(4) where X is O, R¹ at the 5′ position is DMT, MMT or FMOC; R¹ at the3′ position is N,N-diisopropylamino-β-cyanoethoxyphosphine,N,N-diisopropylaminomethoxyphosphine or hydrogen phosphonate; R² is1-propynyl, 3-methyl-1-butynyl, 2-pyrrolyl, 2-thienyl, 2-imidazolyl or2-thiazolyl; and Pr is (H)₂ or diisobutylformamidine.

Preferred Embodiments

One group of preferred oligomers of the present invention can berepresented by the formula (16):

wherein each B, R¹ and R³ are independently selected and have themeanings defined above;

-   -   n is an integer from 0 to 98 (values of 0 to 28 are preferred);        and    -   each X¹ is independently —P(S)(O)—, —P(O)(O)— or —P(CH₃)(O)—,        —P(CH₃)(S)—,    -   provided that at least one B is of the formula (1) or (2) as        defined above; and    -   further provided that when at least one of said nucleomonomers        of said oligomer comprises deoxyuridine 5-substituted by vinyl,        1-butenyl, 1-pentenyl, 1-hexenyl, 1-heptenyl 1-octenyl,        1-propynyl, 1-butynyl, 1-hexynyl, 1-heptynyl, or 1-octynyl, then        the remainder of the nucleomonomers comprising said oligomer are        not solely comprised of phosphodiester linked 2′-deoxyadenosine,        2′-deoxyguanosine, 2′-deoxycytidine, thymidine or a combination        thereof. Methylphosphonate, thionomethylphosphonate or        phosphorothioate substitute linkages enhance the nuclease        stability of the oligomers while their negative impact on        oligomer affinity for target nucleic acids is compensated by the        inclusion of the 5-substituted pyrimidines of the invention.

The most preferred R² group is 1-propynyl. Preferred R³ groups are H,OH, F and O-allyl.

Other preferred oligomers of the invention contain substitute linkagesother than phosphodiester, phosphorothioate, thionomethylphosphonate ormethylphosphonate. Particularly useful forms of these substitutelinkages include riboacetal, formacetal and 3′-thioformacetal linkages,with 3′-thioformacetal being most preferred.

For synthesis of oligomers containing formacetal-type substitutelinkages, in lieu of at least some phosphodiester linkages, dimericsynthons of the formula (6) shown in FIG. 1, wherein the substituents B,X, R¹ and R³ are as defined above are particularly useful.

The foregoing synthon is obtained by first preparing the 5-iodopyrimidine forms of B and then converting these to 5-propynederivatives, for example, by treating the dimer synthon with propyne inthe presence of palladium, CuI, triethylamine, and DMF. These synthonscan be incorporated into an oligomer using standard synthesis techniquesas shown in FIGS. 7, 8, 9 and 11. Synthesis of formacetal and3′-thioformacetal substitute linkages is described in commonly ownedpending U.S. application Ser. No. 07/874,334, filed Apr. 24, 1992, andSer. No. 07/690,786, filed Apr. 24, 1991, which applications areincorporated herein by reference. Trimer synthons containing formacetal,3′-thioformacetal, riboacetal or other substitute linkages are alsopreferred compounds. Trimers and tetramers are preferred for synthesisof oligomers having enhanced permeation across cell membranes.

The synthesis of oligomers containing methylphosphonate andphosphodiester linkages is effected using art-known solid-phase oligomersynthesis techniques. A description of modifications useful in thesynthesis of phosphorothioate linked oligomers are found, for example,in EP publication 288, 163; wherein the oxidation step in solid phaseautomated synthesis using amidite chemistry can be independentlyadjusted at any step to obtain the phosphorothioate. An alternate methodfor synthesis of oligomers with phosphorothioate linkages, via hydrogenphosphonate chemistry, has also been described (Froehler, B., et al.,Nucleic Acid Res (1986) 14: 5399-5467; Froehler, B., Tetrahedron Letters(1986) 27: 5575-5578). Sulfurization can be accomplished using reagentssuch as tetraethylthiuram disulfide, dibenzoyl tetrasulfide,thiophosphoric acid disulfide and the like, 3H-1,2-benzodithiol-3-one1,1-dioxide and the like as described (Vu, H. et al, Tet Lett (1991) 26:3005-3008; Rao, N. V., et al, Tet Lett (1992) 33: 4839-4842; U.S. Pat.No. 5,151,510 issued Sep. 29, 1992; Iyer, R., et al, J Org Chem (1990)55: 4693-4699; Dahl, O. Sulfur Reports (1991) 11: 167-192). Thesesulfurization reagents can be used with either phosphoramidite orhydrogen-phosphonate chemistries. Synthesis of phosphorothioateoligomers having controlled stereochemistry can be used to generatestereoregular invention oligomers as described (InternationalPublication No. EP 0 506 242). The thionomethyl phosphonate is preparedwith methylphosphonamidite followed by sulfurization as described(Roelen, H. P. C. F., et al, Tet Lett (1992) 33: 2357-2360) or with thesulfurization reagents described above.

Covalent Bonding Moiety

Included in some of the oligomers of the invention is a moiety which iscapable of effecting at least one covalent bond between the oligomer andthe target sequence. Multiple covalent bonds can also be formed byproviding a multiplicity of such moieties. The covalent bond ispreferably to a base residue in the target strand, but can also be madewith other portions of the target, including the sugar orphosphodiester. The reaction nature of the moiety which effectscrosslinking determines the nature of the target in the duplex.Preferred crosslinking moieties include acylating and alkylating agents,and, in particular, those positioned relative to the sequencespecificity-conferring portion so as to permit reaction with the targetlocation in the strand.

The crosslinking moiety can conveniently be placed as an analogouspyrimidine or purine residue in the sequence of the oligomer. Theplacement can be at the 5′- and/or 3′-ends, the internal portions of thesequence, or combinations of the above. Placement at the termini topermit enhanced flexibility is preferred. Analogous moieties can also beattached to peptide backbones.

In one preferred embodiment of the invention, a switchback oligomercontaining crosslinking moieties at either end can be used to bridge thestrands of the duplex with at least two covalent bonds. In addition,oligomer sequences of inverted polarity can be arranged in tandem with amultiplicity of crosslinking moieties to strengthen the complex.

Exemplary of alkylating moieties that are useful in the inventioninclude N⁴,N⁴-ethanocytosine and N⁶,N⁶-ethanoadenine.

It is clear that the base need not be a purine or pyrimidine; indeed themoiety to which the reactive function is attached need not be a base atall. Any means of attaching the reactive group is satisfactory so longas the positioning is correct.

Inverted Polarity

In their most general form, inverted polarity oligomers, that canincorporate one or more nucleomonomers described above, contain at leastone segment along their length of the formula:3′---→5′--C--5′-----3′  (1)

-   -   or        5′---→3′--C--3′-----5′  (2)        where —C— symbolizes any method of coupling the nucleomonomer        sequences of opposite polarity (Froehler, B. C., et al        Biochemistry (1992) 31: 1603-1609; Horne, D. A., et al J Am Chem        Soc (1990) 112: 2435-2437; Beal, P. A., et al J Am Chem        Soc (1992) 114: 4976-4978).

In these formulas, the symbol 3′----5′ indicates a stretch of oligomerin which the linkages are consistently formed between the 5′-hydroxyl ofthe ribosyl residue of the nucleomonomer to the left with the 3′- (or2′- for oligomers having 2′, 5′ linkages) hydroxyl of the ribosylresidue of the nucleomonomer to the right (i.e., a region of uniformpolarity), thus leaving the 5′-hydroxyl of the rightmost nucleomonomerribosyl residue free for additional conjugation. Analogously, 5----3′indicates a stretch of oligomer in the opposite orientation wherein thelinkages are formed between the 3′-hydroxyl of the ribosyl residue ofthe left nucleomonomer and the 5′-hydroxyl of the ribosyl residue of thenucleomonomer on the right, thus leaving the 3′-hydroxyl of therightmost nucleomonomer ribosyl residue free for additional conjugation.

The linkage, symbolized by —C—, can be formed so as to link the5′-hydroxyls of the adjacent ribosyl residues in formula (1) or the 3′hydroxyls of the adjacent ribosyl residues in formula (2), or the “—C-”linkage can conjugate other portions of the adjacent nucleomonomers soas to link the inverted polarity strands. “—C—” can represent a linkermoiety, or simply a covalent bond.

It should be noted that if the linkage between strands of invertedpolarity involves a sugar residue, either the 3′- or 2′-position can beinvolved in the linkage, and either of these positions can be in eitherR or S configuration. The choice of configuration will in part determinethe geometry of the oligomer in the vicinity of the linkage. Thus, forexample, if adjacent 3′-positions are used to effect a covalent linkage,less severe deformation of the oligomer chain will generally occur ifboth 3′-hydroxyls involved in the linkage are in the conventional Rconfiguration. If they are both in the S configuration, this will resultin a favorable “kink” in the chain.

In addition to the use of standard oligonucleotide synthesis techniquesor other couplings to effect the 5′-5′ or 3′-3′ linkage between ribosylmoieties, alternative approaches to joining the two strands of invertedpolarity can be employed. For example, the two appended bases of theopposing termini of the inverted polarity oligomer sequences can belinked directly or through a linker, or the base of one can be linked tothe sugar moiety of the other. Any suitable method of effecting thelinkage can be employed. The characterizing aspect of the switchbackoligomers of the invention is that they comprise tandem regions ofinverted polarity, so that a region of 3′→5′ polarity is followed by oneof 5′→3′ polarity, or vice versa, or both.

Depending on the manner of coupling the segments with inverted polarity,this coupling can be effected by insertion of a dimer wherein theappropriate 3′-positions of each member of the dimer or the 5′-positionsof each member of the diner are activated for inclusion of the dimer inthe growing chain, or the conventional synthesis can be continued usingthe condensing nucleomonomer which is blocked in the inverse manner tothat which would be employed if the polarity of the chain were to remainthe same. This additional nucleomonomer can also contain a linker moietywhich can be included before or after condensation to extend the chain.

The synthesis of oligomers having modified residues and/or invertedpolarity can be accomplished utilizing standard solid phase synthesismethods.

In general, there are two commonly used solid phase-based approaches tothe synthesis of oligomers containing conventional 3′→5′ or 5′→3′linkages, one involving intermediate phosphoramidites and the otherinvolving intermediate phosphonate linkages. In the phosphoramiditebased synthesis, a suitably protected nucleomonomer having acyanoethylphosphoramidite at the position to be coupled is reacted withthe free hydroxyl of a growing nucleomonomer chain derivatized to asolid support. The reaction yields a cyanoethylphosphite, which linkagemust be oxidized to the cyanoethylphosphate at each intermediate step,since the reduced form is unstable to acid. The H-phosphonate-basedsynthesis is conducted by the reaction of a suitably protectednucleomonomer containing an H-phosphonate moiety at a position to becoupled with a solid phase-derivatized nucleomonomer chain having a freehydroxyl group, in the presence of a suitable activator to obtain anH-phosphonate diester linkage, which is stable to acid. Thus, theoxidation to the phosphate or thiophosphate can be conducted at anypoint during the synthesis of the oligomer or after synthesis of theoligomer is complete. The H-phosphonates can also be converted tophosphoramidate derivatives by reaction with a primary or secondaryamine in the presence of carbon tetrachloride. To indicate the twoapproaches generically, the incoming nucleomonomer is regarded as havinga “coupling phosphite/phosphate” group.

Variations in the type of substitute linkage are achieved by, forexample, using the methyl phosphonate precursors rather than theH-phosphonates per se, using thiol derivatives of the nucleomonomermoieties and generally by methods known in the art. Nonphosphorous basedlinkages can also be used, such as the formacetal 3′-thioformacetal,3′-amino and 5′-ether type linkages described above.

Thus, to obtain an oligomer segment which has a 3′→5′ polarity, anucleomonomer protected at the 5′-position and containing a couplingphosphite/phosphate group at the 3′-position is reacted with thehydroxyl at the 5′-position of a nucleomonomer coupled to a solidsupport through its 3′-hydroxyl. The resulting condensed oligomer isdeprotected and the reaction repeated with an additional 5′-protected,3′-phosphite/phosphate coupling nucleomonomer. Conversely, to obtain anoligomeric segment of 5′→3′ polarity, a nucleomonomer protected in the3′-position and containing a coupling phosphite/phosphate in the5′-position is reacted with a oligomer or nucleomonomer attached to asolid support through the 5′-position, leaving the 3′-hydroxyl availableto react. Similarly, after condensation of the incoming nucleomonomer,the 3′-group is deprotected and reacted with an additional 3′-protected,5′-coupling nucleomonomer. The sequence is continued until the desirednumber of nucleomonomers have been added.

This oligomer chain elongation will proceed in conformance with apredetermined sequence in a series of condensations, each one of whichresults in the addition of another nucleomonomer. Prior to the additionof a nucleomonomer having a coupling phosphite/phosphate, the protectinggroup on the solid support-bound nucleomonomer is removed. Typically,for example, removal of the commonly-employed dimethoxytrityl (DMT)group is done by treatment with 2.5% v/v dichloroaceticacid/dichloromethane, although 1% w/v trichloroaceticacid/dichloromethane or ZnBr₂-saturated nitromethane, are also useful.Other deprotection procedures suitable for other protecting groups willbe apparent to those of ordinary skill in the art. The deprotectednucleomonomer or oligomer bound to solid support is then reacted withthe suitably protected nucleomonomer containing a couplingphosphite/phosphate. After each cycle the carrier bound nucleomonomer ispreferably washed with anhydrous pyridine/acetonitrile (1:1, v/v), againdeprotected, and the condensation reaction is completed in as manycycles as are required to form the desired number of congruent polarityinternucleoside bonds which will be converted to phosphoramidates,phosphorodithioates, phosphorothioates or phosphodiesters as desired.

In one embodiment, to provide the switchback linker, the incomingcoupling, protected nucleomonomer is provided in the opposite polarityto the support-bound oligomers. Thus, for example, where thesupport-bound oligomer is 3′→5′, the deprotected 5′-hydroxyl is reactedwith a 3′-protected, 5′-coupling monomer, and the synthesis continuedwith monomers coupled at the 5′-position and protected at the3′-position.

In another embodiment, to provide the switchback linker, a dimer synthoncontaining the linker element having one end which is coupled forcondensation (such as a hydrogen phosphonate) to the support-boundoligomer and another end which is a protected hydroxyl group (orprotected thio group) is condensed onto the support-bound oligomer. Thelinked dimer is condensed and deprotected using the same conditions asthose used to condense and deprotect the protected nucleomonomerhydrogen phosphonate. Subsequent extension of the oligomer chain thenuses nucleomonomer residues which are coupled and protected in theopposite manner from those used to synthesize the previous portion ofthe chain.

One approach to this synthesis, using a linker already derivatized totwo nucleomonomer residues which will be included in each portion of thestrand is illustrated in FIG. 5. The 5′→3′ nucleomonomer portion of thestrand is coupled using the 3′-DMT-5′-coupling phosphate nucleomonomers,as conventionally, to solid support. The switchback linker isderivatized to two nucleomonomer residues through their 3′-positions;the remaining 5′-positions are derivatized by the protecting group DMTin one nucleomonomer residue and a phosphonate residue in the other. Thederivatized linker is coupled to the solid supported strand understandard reagent conditions and then deprotected conventionally. Furtherstandard nucleomonomer coupling results in extension of the chain in the3′→5′ orientation.

A particularly preferred dimer synthon used to mediate the switchback inan oligomer is the O-xyloso linker (compound 9 and Formula (21) in FIG.5. The O-xyloso linker consists of two xylose-nucleomonomers (18) linkedto each other by o-xylene at the 3′-position of each xylose sugar. Theswitchback linker synthon was synthesized using α,α′-orthodibromoxyleneand 5′-DMT nucleomonomer (18) to give the dimer (19) as shown in FIG. 5.The dimer was converted to the H-phosphonate (21) and was used in solidphase synthesis to generate oligomers. Linkers containing the bases (atposition “B” in FIG. 5) thymine, 5-methylcytosine,8-hydroxy-N⁴-methyladenine, pseudoisocytosine, 5-propynyluracil orcytosine are synthesized as homodimers. However, the switchback linkerdimers can also be synthesized as mixed heterodimers that are separatedchromatographically.

A particularly useful synthon in the preparation of oligomers containinginverted polarity is of formula (5) shown in FIG. 1, wherein each Y andeach B is independently chosen and have the meanings previously defined,and wherein one or both of the bases, B, can optionally be the modifiedbase residues of formula (1) or (2) of the invention.

2′ Modified Oligomers

Included in some of the oligomers containing C-5 modified pyrimidines ofthe invention are modifications of the ribose or deoxyribose sugar.2′-O-methyl-, 2′-O-ethyl- and 2′-O-allyl oligomers have been synthesizedand shown to bind to single-stranded complementary nucleic acidsequences (Cotten, M., et al., Nucleic Acids Res (1990) 19: 2629-2635;Blencowe, B. J., et al., Cell (1989) 59: 531-539; Sproat, B. S., et al.,Nucleic Acids Res (1989) 17: 3373-3386; Inoue, H., et al., Nucleic AcidsRes (1987) 15: 6131-6148; Morisawa, H., et al., European Patent SerialNo. 0339842; Chavis, C., et al., J Organic Chem (1982) 47: 202-206;Sproat, B. S., et al, Nucleic Acids Res (1991) 19: 733-738). The2′-modified oligomers were reported to be relatively nuclease stablecompared to unmodified controls. Synthesis of 2′ fluoro nucleomonomersand their incorporation into oligomers has also been described(Codington, J. F., et al, J org Chem (1964) 29: 558-564; Fazakerley, G.V., et al, FEBS Lett (1985) 182: 365-369). Synthesis of oligomer analogscontaining the modified bases described herein would be based on methodsdescribed. Synthesis of oligomers containing 2′-amino nucleomonomers hasbeen described (Pieken, W. A., et al, Science (1991) 253: 314-317).

In an additional use of bases (1) and (2) in oligomers of the invention,2′-O-allyl modified sugar forms of the nucleomonomers containing the5-substituted bases (1) and (2) of the invention are included in theoligomer. Other 2′-O-allyl-substituted nucleomonomers can also be usedat other positions in the oligomer. The 2′-O-allyl nucleomonomers can beprepared using standard methods; set forth below is a method forsynthesis of the 2′-O-allyl derivatized nucleomonomers containing theinvention pyrimidines through a common intermediate. Thus, for example,5-(1-propynyl)uridine is first protected at the 5′- and 3′-positionsusing a tetraisopropyldisiloxane reagent, and then at the 4-oxy positionusing ortho-nitrophenol. The protected nucleoside is then converted tothe 2′-O-allyl derivative with allyl ethyl carbonate; this usefulintermediate is alternatively converted to the 2′-o-allyl-derivatized5-(1-propynyl)uridine or the corresponding 5-(1-propynyl)cytidine. Thesequence of reactions for this conversion are outlined in FIG. 4.

The nucleomonomers derivatized at the 2′-position can be incorporatedinto oligomers in the same manner as underivatized forms.

Synthesis of 2′-thioalkyl nucleomonomers is accomplished as described inFIG. 6. The protocol is useful for synthesis of 2′-thioalkyl pyrimidinesvia formation of an anhydro intermediate 7 that is subsequentlyconverted to thioalkyl nucleomonomer (22). The group designated W isdefined as lower alkane (methyl, ethyl, propyl, isopropyl, butyl orisobutyl) or lower alkene including allyl. The protocol was used tosynthesize 5′-DMT blocked 5-methyluridine 3′-H-phosphonate. The startingmaterial 6 was obtained from 5-methyluridine (Markiewicz, W. T., J.Chem. Res (M) (1979) 0181-0197. Alternate blocking groups at the 5′- and3′-positions, such as tetrahydropyran can also be utilized to obtain anequivalent starting material. The method shown in FIG. 6 can thus beused to synthesize 2′-thioalkyl derivatives of the nucleomonomerscontaining the modified bases of the present invention in addition tosynthesis of other modified pyrimidine nucleomonomers such as2′-thioalkylcytidine, 2′-thioalkylthymidine,2′-thioalkyl-N⁴-N⁴-ethanocytidine or 2′-thioalkyluridine. Conversion ofthe nucleomonomer (22) to other 5′- and 3′-derivatized compounds such asMMT, β-cyanoethylphosphoramidite, or methylphosphoramidite-blockednucleomonomers can easily be accomplished using appropriate reagents.

Dimer and Trimer Synthons for Oligomers Containing Substitute Linkages

Oligomers containing substitute linkages that link adjacentnucleomonomer analog residues are preferably synthesized using suitablyblocked dimer synthons as a starting material. For diners wherein one orboth base residues are 5-R²-U or 5-R²—C or related analogs, synthesis ofa formacetal or 3′-thioformacetal-linked diner is accomplished asdescribed herein. An exemplary dimer containing a formacetal linkage offormula (6) shown in FIG. 1, Y, X, B and R³ are as defined herein.

FIGS. 7 and 8 show synthesis schemes for intermediates in oligomersynthesis. In both Figures, the structure U-I represents 5-iodouraciland U—≡—CH₃ represents 5-(1-propynyl)uracil. Synthesis of a3′-thioformacetal dimer or a trimer can conveniently be accomplished. Asshown in FIG. 7, a 5-iodouridine nucleomonomer, (25), protected at the3′-position by esterification is first reacted with paraformaldehyde inthe presence of HCl to obtain the derivatized nucleomonomer, (26),containing the substituent ClCHO₂— at the 5′-position. The nucleomonomercan be esterified using, for example, a long-chain alkyl or aromaticacid, such a decyl, hexyl, benzoyl, or phenoxyacetyl. In this firststep, the 3′-esterified nucleomonomer is treated with an excess ofparaformaldehyde in an inert solvent at low temperature and anhydrousHCl is bubbled through the reaction mixture. The solution isconveniently dried and the solvent removed to obtain the intermediate.

The intermediate, (26), shown as the chloro-methylether (ClCH₂O—) at the5′-position of the nucleo-side, is then dissolved in an inert solvent. Asolution of a second nucleomonomer (5-(1-propynyl)uridine derivative),10, protected at the 5′-position, for example by DMT, and bearing an —SHsubstituent at the 3′-position along with a base, preferablydiisopropylethylamine (DIPEA), in an inert solvent, is also prepared.The chloromethyl ether intermediate is added dropwise to this solutionand the reaction mixture is stirred for several hours.

The reaction is washed with water, and the organic layer is separatedand dried to obtain the dimerized product having the 3′-SCH₂O-5′ linkageand protected at the 5′- and 3′-positions, as above. The resulting dimeris deprotected at the 3′-position and then converted to the propynederivative as shown and described in Example 1. Where the diner is to beused in standard oligomer synthesis, it is converted to the hydrogenphosphonate using 2-chloro-4H-1,2,3-benzodioxaphosphorin-4-one (vanBoom's reagent for phosphytylation (PA)). FIG. 8 shows the synthesis ofa 3′-thioformacetal trimer.

Synthesis of riboacetal linked dimers is shown in FIG. 9. 5′-DMT,3′-H-phosphonate diners can be directly utilized for incorporation intooligomers by conventional methods or an appropriate precursor can beutilized as needed for conversion to trimer, tetramer or longer lengtholigomers.

Synthesis of Ethynyl Heteroaryl and Heteroaryl Derivatized Bases

FIGS. 14, 15 and 16 show synthetic schemes for synthesis ofnucleomonomers having invention bases with ethynyl heteroaryl orheteroaryl groups at the 5 position. The nucleomonomers can be convertedto blocked monomers suitable for incorporation into oligomers byconventional methods.

Synthesis of 5-phenyl-2′-deoxyuridine was accomplished as previouslydescribed using phenyltrimethylstannane (Crisp, G., et al., TetrahedronLetters (1990) 31: 1347-1350). An analogous protocol usingpyridinyltrimethylstannane or pyridinyltributylstannane or the like as astarting material which is obtained from bromopyridine is used tosynthesize 5-(2-pyridinyl)-2′-deoxyuridine (Example 15). Synthesis ofheteroarylstannanes is conveniently accomplished as described (Bailey,T. R. Tet Lett (1986) 27: 4407-4410; Jutzi, P. et al, J. organometalChem. (1983) 246: 163-168; Molloy, K. C. et al, J. Organometal Chem.(1989) 365: 61-73).

Synthesis of 5-substituted pyrimidine nucleomonomers with heteroarylgroups such as 2-thiazoyl, 1-methyl-2-imidazolyl, 2-oxazoyl, 2-furanyland the like can be accomplished using a published protocol (Wigerinck,P. et al., J Med Chem (1991) 34: 2383-2389; Peters, D., et al,Nucleosides and Nucleotides (1992) 11: 1151-1173) followed by conversionto the corresponding nucleomonomer by standard methods (see Example 16).The 5-cyano substituent is prepared as described (Inoue, T., et al, ChemPharm Bull (1978) 9: 2657-2663) and also can be used as a startingelectrophile to build heteroaryl substituted nucleomonomers as described(Wigerinck, P., et al, J Med Chem (1991) 34: 1767-1772).

Ethynyl heteroaryl derivatives are prepared from ethynyltrimethylsilaneand an appropriate heteroaryl as described (Austin, W. B., et al, J OrgChem (1981) 46: 2280-2286) (see Example 14). The deprotected ethynyl isthen introduced into the nucleomonomer by standard procedures (Examples1 and 14).

Utility and Administration

As the oligomers of the invention are capable of significantsingle-stranded or double-stranded target nucleic acid binding activityto form duplexes, triplexes or other forms of stable association, theseoligomers are useful in diagnosis and therapy of diseases that areassociated with expression of one or more genes such as those associatedwith many different pathological conditions. Therapeutic applicationscan employ the oligomers to specifically inhibit the expression of genes(or inhibit translation of RNA sequences encoded by those genes) thatare associated with either the establishment or the maintenance of apathological condition. Exemplary genes or RNAs encoded by those genesthat can be targeted include those that encode enzymes, hormones, serumproteins, adhesion molecules, receptor molecules, cytokines, oncogenes,growth factors, and interleukins. Target genes or RNAs can be associatedwith any pathological condition such as those associated withinflammatory conditions, cardiovascular disorders, immune reactions,cancer, viral infections, bacterial infections and the like.

Oligomers of the present invention are suitable for both in vivo and Avivo therapeutic applications. Indications for ex vivo uses includetreatment of cells such as bone marrow or peripheral blood in conditionssuch as leukemia or viral infection. Target genes or RNAs encoded bythose genes that can serve as targets for cancer treatments includeoncogenes, such as ras, k-ras, bcl-2, c-myb, bcr, c-myc, c-abl oroverexpressed sequences such as mdm2, oncostatin M, IL-6 (Kaposi'ssarcoma), HER-2 and translocations such as bcr/abl. Viral gene sequencesor RNAs encoded by those genes such as polymerase or reversetranscriptase genes of CMV, HSV-1, HSV-2, HTLV-1, HIV-1, HIV-2, HBV,HPV, VZV, influenza virus, rhinovirus and the like are also suitabletargets. Application of specifically binding oligomers can be used inconjunction with other therapeutic treatments. Other therapeuticindications for oligomers of the invention include (1) modulation ofinflammatory responses by modulating expression of genes such as IL-1receptor, IL-1, ICAM-1 or E-Selectin that play a role in mediatinginflammation and (2) modulation of cellular proliferation in conditionssuch as arterial occlusion (restenosis) after angioplasty by modulatingthe expression of (a) growth or mitogenic factors such as non-musclemyosin, myc, fos, PCNA, PDGF or FGF or their receptors, or (b) cellproliferation factors such as c-myb. Other suitable extracellularproliferation factors such as TGFα, IL-6, γINF, protein kinase C may betargeted for treatment of psoriasis or other conditions. In addition,EGF receptor, TGFα or MHC alleles may be targeted in autoimmunediseases.

Delivery of oligomers of the invention into cells can be enhanced by anysuitable method including calcium phosphate, DMSO, glycerol or dextrantransfection, electroporation or by the use of cationic anionic and/orneutral lipid compositions or liposomes by methods described(International Publication Nos. WO 90/14074, WO 91/16024, WO 91/17424,U.S. Pat. No. 4,897,355). The oligomers can be introduced into cells bycomplexation with cationic lipids such as DOTMA (which can or can notform liposomes) which complex is then contacted with the cells. Suitablecationic lipids include but are not limited toN-(2,3-di(9-(Z)-octadecenyloxyl))-prop-1-yl-N,N,N-trimethylammonium(DOTMA) and its salts,1-O-oleyl-2-O-oleyl-3-dimethylaminopropyl-β-hydroxyethylammonium and itssalts and 1,2-bis(oleyloxy)-3-(trimethylammonio) propane and its salts.

Enhanced delivery of the invention oligomers can also be mediated by theuse of (i) viruses such as Sendai virus (Bartzatt, R., Biotechnol ApplBiochem (1989) 11: 133-135) or adenovirus (Wagner, E., et al, Proc NatlAcad Sci (1992) 89: 6099-6013; (ii) polyamine or polycation conjugatesusing compounds such as polylysine, protamine or N1,N12-bis(ethyl)spermine (Wagner, E., et al, Proc Natl Acad Sci (1991) 88:4255-4259; Zenke, M., et al, Proc Natl Acad Sci (1990) 87: 3655-3659;Chank, B. K., et al, Biochem Biophys Res Commun (1988) 157: 264-270;U.S. Pat. No. 5,138,045); (iii) lipopolyamine complexes using compoundssuch as lipospermine (Behr, J.-P., et al, Proc Natl Acad Sci (1989) 86:6982-6986; Loeffler, J. P., et al J Neurochem (1990) 54: 1812-1815);(iv) anionic, neutral or pH sensitive lipids using compounds includinganionic phospholipids such as phosphatidyl glycerol, cardiolipin,phosphatidic acid or phosphatidylethanolamine (Lee, K.-D., et al,Biochim Biophys ACTA (1992) 1103: 185-197; Cheddar, G., et al, ArchBiochem Biophys (1992) 294: 188-192; Yoshimura, T., et al, Biochem Int(1990) 20: 697-706); (v) conjugates with compounds such as transferrinor biotin or (vi) conjugates with compounds such as serum proteins(including albumin or antibodies), glycoproteins or polymers (includingpolyethylene glycol) that enhance pharmacokinetic properties ofoligomers in a subject. As used herein, transfection refers to anymethod that is suitable for delivery of oligomers into cells. Anyreagent such as a lipid or any agent such as a virus that can be used intransfection protocols is collectively referred to herein as a“permeation enhancing agent”. Delivery of the oligomers into cells canbe via cotransfection with other nucleic acids such as (i) expressableDNA fragments encoding a protein(s) or a protein fragment or (ii)translatable RNAs that encode a protein(s) or a protein fragment.

The oligomers can thus be incorporated into any suitable formulationthat enhances delivery of the oligomers into cells. Suitablepharmaceutical formulations also include those commonly used inapplications where compounds are delivered into cells or tissues bytopical administration. Compounds such as polyethylene glycol, propyleneglycol, azone, nonoxonyl-9, oleic acid, DMSO, polyamines orlipopolyamines can be used in topical preparations that contain theoligomers.

The invention oligomers can be conveniently used as reagents forresearch or production purposes where inhibition of gene expression isdesired. There are currently very few reagents available thatefficiently and specifically inhibit the expression of a target gene byany mechanism. Oligomers that have been previously reported to inhibittarget gene expression frequently have nonspecific effects and/or do notreduce target gene expression to very low levels (less than about 40% ofuninhibited levels). The invention oligomers are, by comparison,extremely potent with IC₅₀ values as low as 0.05 μM in microinjectionassays (Example 6, Table 4 below). These levels of potency permitapplication of the oligomers to cells with efficient inhibition oftarget gene expression while avoiding significant nonspecific effects onthe cell. In view of this, it is clear that the invention oligomersrepresent a unique class of reagents that can be used to probe genefunction and to probe the role of single stranded or double strandednucleic acids.

Thus, the results as described herein provide a method of inhibitingexpression of a selected protein or proteins in a subject or in cellswherein the proteins are encoded by DNA sequences and the proteins aretranslated from RNA sequences, comprising the steps of: introducing anoligomer of the invention into the cells; and permitting the oligomer toform a triplex with the DNA or RNA or a duplex with the DNA or RNAwhereby expression of the protein or proteins is inhibited. The methodsand oligomers of the present invention are suitable for modulating geneexpression in both procaryotic and eucaryotic cells such as bacterial,parasite, yeast and mammalian cells.

The results described below (Example 6 and 7) demonstrate that theoligomers, when used to inhibit gene expression by an antisensemechanism, can be RNase H “competent” or RNase H “incompetent” species.Oligomers having modifications such as 2′-substitutions (2′-O-allyl andthe like) or certain uncharged linkages (methylphosphonate,phosphoramidate and the like) are usually incompetent as a substratethat is recognized by and/or acted on by RNase H. RNase H competence canfacilitate antisense oligomer function by degrading the target RNA in anRNA-oligomer duplex (Dagle, J. M., et al, Nucl Acids Res (1990) 18:4751-4757; Walder, J. A. et al, International Publication Number WO89/05358). The enzyme cleaves RNA in RNA-DNA duplexes.

In order to retain RNase H competence, an oligomer requires a RNase Hcompetent domain of three or more competent contiguous linkages locatedwithin it (Quartin, R. S., et al, Nucl Acids Res (1989) 17: 7253-7262).Design of oligomers resistant to nuclease digestion will have terminallinkage, sugar and/or base modifications to effect nuclease resistance.Thus, the oligomers can be designed to have modified nucleomonomerresidues at either or both the 5′- and/or 3′-ends, while having aninternal RNase H competent domain.

Exemplary oligomers that retain RNase H competence would generally haveuniform polarity and would comprise about 2 to about 12 nucleomonomersat the 5′-end and at the 3′-end which stabilize the oligomer to nucleasedegradation and about three to about 26 nucleomonomers that function asa RNase H competent domain between the RNase H incompetent 3′- and5′-ends. Variations on such an oligomer would include (1) a shorterRNase H competent domain comprising 1 or 2 RNase H competent linkages,(2) a longer RNase H incompetent domain comprising up to 15, 20 or moresubstitute linkages or nucleomonomers, (3) a longer RNase H competentdomain comprising up to 30, 40 or more linkages, (4) oligomers with onlya single RNase H incompetent domain at the 3′ end or at the 5′ end, or(5) oligomers having more than one RNase H competent domain. RNase Hcompetence also applies as a consideration to oligomers having one ormore regions of inverted polarity, to circular oligomers and to othertypes of oligomers.

Oligomers containing as few as about 8 nucleomonomers can be used toeffect inhibition of target protein(s) expression by formation of duplexor triplex structures with target nucleic acid sequences. However,linear oligomers used to inhibit target protein expression via duplex ortriplex formation will preferably have from about 12 to about 20nucleomonomer residues.

Oligomers containing bases of the invention can be convenientlycircularized as described (International Publication No. WO 92/19732;Kool, E. T. J Am Chem Soc (1991) 113: 6265-6266; Prakash, G., et al. JAm Chem Soc (1992) 114: 3523-3527). Such oligomers are suitable forbinding to single-stranded or double-stranded nucleic acid targets.Circular oligomers can be of various sizes. Such oligomers in a sizerange of about 22-50 nucleomonomers can be conveniently prepared. Thecircular oligomers can have from about three to about six nucleomonomerresidues in the loop region that separate binding domains of theoligomer as described (Prakash, G. ibid). Oligomers can be enzymaticallycircularized through a terminal phosphate by ligase or by chemical meansvia linkage through the 5′- and 3′-terminal sugars and/or bases.

The oligomers can be utilized to modulate target gene expression byinhibiting the interaction of nucleic acid binding proteins responsiblefor modulating transcription (Maher, L. J., et al, Science (1989) 245:725-730) or translation (Example 7 below). The oligomers are thussuitable as sequence-specific agents that compete with nucleic acidbinding proteins (including ribosomes, RNA polymerases, DNA polymerases,translational initiation factors, transcription factors that eitherincrease or decrease transcription, protein-hormone transcriptionfactors and the like). Appropriately designed oligomers can thus be usedto increase target protein synthesis through mechanisms such as bindingto or near a regulatory site that transcription factors use to repressexpression or by inhibiting the expression of a selected repressorprotein itself.

The invention oligomers can be designed to contain secondary or tertiarystructures, such as pseudoknots or pseudo-half-knots (Ecker, D. J., etal, Science (1992) 257: 958-961). Such structures can have a more stablesecondary or tertiary structure than corresponding unmodified oligomers.The enhanced stability of such structures would rely on the increasedbinding affinity between regions of self complementarity in a singleoligomer or regions of complementarity between two or more oligomersthat form a given structure. Such structures can be used to mimicstructures such as the HIV TAR structure in order to interfere withbinding by the HIV Tat protein (a protein that binds to TAR). A similarapproach can be utilized with other transcription or translation factorsthat recognize higher nucleic acid structures such as stems, loops,hairpins, knots and the like. Alternatively, the invention oligomers canbe used to (1) disrupt or (2) bind to such structures as a method to (1)interfere with or (2) enhance the binding of proteins to nucleic acidstructures.

In addition to their use in antisense or triple helix therapies, theoligomers of the invention can also be applied as therapeutic ordiagnostic agents that function by direct displacement of one strand ina duplex nucleic acid. Displacement of a strand in a natural duplex suchas chromosomal DNA or duplex viral DNA, RNA or hybrid DNA/RNA ispossible for oligomers with a high binding affinity for theircomplementary target sequences. Therapeutic applications of oligomers bythis method of use, referred to herein as D-looping or “D-loop therapy”has not previously been possible because the affinity of natural DNA orRNA for its complementary sequence is not great enough to efficientlydisplace a DNA or RNA strand in a duplex. Therapeutic efficacy ofoligomers that function by D-looping would result from high affinitybinding to a complementary sequence that results in modulation of thenormal biological function associated with the nucleic acid target.Types of target nucleic acids include but are not limited to (i) genesequences including exons, introns, exon/intron junctions,promoter/enhancer regions and 5′ or 3′ untranslated regions, (ii)regions of nucleic acids that utilize secondary structure in order tofunction (e.g. the HIV TAR stem-loop element or tRNAs), (iii) nucleicacids that serve structural functions such as telomeres or centromeresand (iv) any other duplex region. It is clear that oligomers can besynthesized with discrete functional domains wherein one region of anoligomer binds to a target by D-looping while an adjacent region binds atarget molecule by say, forming a triple helix or binding as an aptamerto a protein. Alternatively, a D-looping oligomer can bind to eachstrand in a duplex by switching the strand to which the oligomer binds(i.e. by having one region of the oligomer that binds to one is strandand another region that binds to the complementary strand). Thecontrolling elements that dictate the mode of binding (i.e. triple helixor D-loop) are the sequence of the oligomer and the inherent affinitybuilt into the oligomer. Base recognition rules in Watson-Crick duplexbinding differ from those in Hoogsteen controlled triplex binding.Because of this, the oligomer base sequence can be used to dictate thetype of binding rules an oligomer will utilize.

D-loop structures are formed in nature by enzyme-mediated processes(Harris, L. D. et al., J Biol Chem (1987) 262: 9285-9292) or areassociated with regions where DNA replication occurs (Jacobs, H. T. etal., Nucl Acids Res (1989) 17: 8949-8966). D-loops that arise from thebinding of oligomers can result from a one or two step process. Directdisplacement of a target strand will give rise to a D-loop by a singlebinding event. However, D-looping can also occur by forming a triplehelix which facilitates a strand displacement event leading to theD-loop.

Ribozymes containing bases of the invention can be designed in order todesign species with altered characteristics. Ribozymes that cleavesingle stranded RNA or DNA (Robertson, D. L., et al Nature (1990) 344:467-468) have been described. Therapeutic applications for ribozymeshave been postulated (Sarver, N. et al, Science (1990) 247: 1222-1225;International Publication Number WO 91/04319). Secondary or tertiarystructure necessary for ribozyme function can be affected by design ofappropriate oligomer sequences. For example, ribozymes having targetingdomains containing bases of the invention will have higher affinity,while maintaining base pairing specificity, for target sequences.Because of the higher affinity of the invention bases for theircomplementary sequences, shorter recognition domains in a ribozyme (anadvantage in manufacturing) can be designed which lead to more favorablesubstrate turnover (an advantage in ribozyme function).

In therapeutic applications, the oligomers are utilized in a mannerappropriate for treatment of a variety of conditions by inhibitingexpression of appropriate target genes. For such therapy, the oligomerscan be formulated for a variety of modes of administration, includingsystemic, topical or localized administration. Techniques andformulations generally can be found in Remington's Pharmaceuticalsciences, Mack Publishing Co., Easton, Pa., latest edition. The oligomeractive ingredient is generally combined with a carrier such as a diluentor excipient which can include fillers, extenders, binders, wettingagents, disintegrants, surface-active agents, or lubricants, dependingon the nature of the mode of administration and dosage forms. Typicaldosage forms include tablets, powders, liquid preparations includingsuspensions, emulsions and solutions, granules, capsules andsuppositories, as well as liquid preparations for injections, includingliposome preparations.

For systemic administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the oligomers of the invention are formulated in liquidsolutions, preferably in physiologically compatible buffers such asHank's solution or Ringer's solution. In addition, the oligomers can beformulated in solid form and redissolved or suspended immediately priorto use. Lyophilized forms are also included. Dosages that can be usedfor systemic administration preferably range from about 0.01 mg/Kg to 50mg/Kg administered once or twice per day. However, different dosingschedules can be utilized depending on (i) the potency of an individualoligomer at inhibiting the activity of its target DNA or RNA, (ii) theseverity or extent of a pathological disease state associated with agiven target gene, or (iii) the pharmacokinetic behavior of a givenoligomer.

Systemic administration can also be by transmucosal or transdermalmeans, or the compounds can be administered orally. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, bile salts and fusidic acidderivatives for transmucosal administration. In addition, detergents canbe used to facilitate permeation. Transmucosal administration can bethrough use of nasal sprays, for example, or suppositories. For oraladministration, the oligomers are formulated into conventional oraladministration forms such as capsules, tablets, and tonics.

For topical administration, the oligomers of the invention areformulated into ointments, salves, gels, or creams, as is generallyknown in the art. Formulation of the invention oligomers for ocularindications such as viral infections would be based on standardcompositions known in the art.

In addition to use in therapy, the oligomers of the invention can beused as diagnostic reagents to detect the presence or absence of thetarget nucleic acid sequences to which they specifically bind. Theenhanced binding affinity of the invention oligomers is an advantage fortheir use as primers and probes. Diagnostic tests cab be conducted byhybridization through either double or triple helix formation which isthen detected by conventional means. For example, the oligomers can belabeled using radioactive, fluorescent, or chromogenic labels and thepresence of label bound to solid support detected. Alternatively, thepresence of a double or triple helix can be detected by antibodies whichspecifically recognize these forms. Means for conducting assays usingsuch oligomers as probes are generally known.

The use of oligomers containing the modified bases as diagnostic agentsby triple helix formation is advantageous since triple helices formunder mild conditions and the assays can thus be carried out withoutsubjecting test specimens to harsh conditions. Diagnostic assays basedon detection of RNA for identification of bacteria, fungi or protozoasequences often require isolation of RNA from samples or organisms grownin the laboratory, which is laborious and time consuming, as RNA isextremely sensitive to ubiquitous nucleases.

The oligomer probes can also incorporate additional modifications suchas modified sugars and/or substitute linkages that render the oligomerespecially nuclease stable, and would thus be useful for assaysconducted in the presence of cell or tissue extracts which normallycontain nuclease activity. Oligomers containing terminal modificationsoften retain their capacity to bind to complementary sequences withoutloss of specificity (Uhlmann et al., Chemical Reviews (1990) 90:543-584). As set forth above, the invention probes can also containlinkers that permit specific binding to alternate DNA strands byincorporating a linker that permits such binding (Froehler, B. C., etal, Biochemistry (1992) 31: 1603-1609); Horne et al., J Am Chem Soc(1990) 112: 2435-2437).

Incorporation of base analogs of the present invention into probes thatalso contain covalent crosslinking agents has the potential to increasesensitivity and reduce background in diagnostic or detection assays. Inaddition, the use of crosslinking agents will permit novel assaymodifications such as (1) the use of the crosslink to increase probediscrimination, (2) incorporation of a denaturing wash step to reducebackground and (3) carrying out hybridization and crosslinking at ornear the melting temperature of the hybrid to reduce secondary structurein the target and to increase probe specificity. Modifications ofhybridization conditions have been previously described (Gamper et al.,Nucleic Acids Res (1986) 14: 9943).

Oligomers of the invention are suitable for use in diagnostic assaysthat employ methods wherein either the oligomer or nucleic acid to bedetected are covalently attached to a solid support as described (U.S.Pat. No. 4,775,619). The oligomers are also suitable for use indiagnostic assays that rely on polymerase chain reaction techniques toamplify target sequences according to described methods (European PatentPublication No. 0 393 744). Oligomers of the invention containing5-modified pyrimidines are compatible with polymerases used inpolymerase chain reaction methods such as the Taq or Vent™ polymerase.Oligomers of the invention can thus be utilized as primers in PCRprotocols or triphosphate pyrimidine monomers having R² at the5-position can be utilized as a substrate by DNA or RNA polymerasesderived from thermophiles (Taq or Vent™) or other sources (E. coli,human, retrovirus, etc) to generate the oligomers of the invention invarious diagnostic protocols. Synthesis of monomer triphosphates isaccomplished by known methods (Otvos, L., et al, Nucl Acids Res (1987)15: 1763-1777).

The oligomers are useful as primers that are discrete sequences or asprimers with a random sequence. Random sequence primers are generallyabout 6 or 7 nucleomonomers in length. Such primers can be used invarious nucleic acid amplification protocols (PCR, ligase chainreaction, etc) or in cloning protocols. The 5-substitutions of theinvention generally do not interfere with the capacity of the oligomerto function as a primer. Oligomers of the invention having2′-modifications at sites other than the 3′ terminal residue, othermodifications that render the oligomer RNase H incompetent or otherwisenuclease stable can be advantageously used as probes or primers for RNAor DNA sequences in cellular extracts or other solutions that containnucleases. Thus, the oligomers can be used in protocols for amplifyingnucleic acid in a sample by mixing the oligomer with a sample containingtarget nucleic acid, followed by hybridization of the oligomer with thetarget nucleic acid and amplifying the target nucleic acid by PCR, LCRor other suitable methods.

The oligomers derivatized to chelating agents such as EDTA, DTPA oranalogs of 1,2-diaminocyclohexane acetic acid can be utilized in variousin vitro diagnostic assays as described (U.S. Pat. Nos. 4,772,548,4,707,440 and 4,707,352). Alternatively, oligomers of the invention canbe derivatized with crosslinking agents such as5-(3-iodoacetamidoprop-1-yl)-2′-deoxyuridine or5-(3-(4-bromobutyramido)prop-1-yl)-2′-deoxyuridine and used in variousassay methods or kits as described (International Publication No. WO90/14353).

In addition to the foregoing uses, the ability of the oligomers toinhibit gene expression can be verified in in vitro systems by measuringthe levels of expression in subject cells or in recombinant systems, byany suitable method (Graessmann, M., et al., Nucleic Acids Res (1991)19: 53-59).

All references cited herein are incorporated herein by reference intheir entirety.

The following examples are intended to illustrate, but not to limit, theinvention. Efforts have been made to insure accuracy with respect tonumbers used (e.g., amounts, temperatures, etc.), but some experimentalerrors and deviations should be taken into account. Unless indicatedotherwise, parts are parts by weight, temperature is in degreesCentigrade, and pressure is at or near atmospheric.

EXAMPLE 1 Synthesis of 5-(1-Propynyl)-2′-Deoxyuridine H-PhosphonateMonomer and Oligomers Containing the Analog

In a 50 mL round bottom flask is placed:

-   -   a) 708 mg (2 mmole) 5-iodo dU    -   b) 10 mL anhydrous DMF    -   c) 76 mg (0.4 mmole) CuI    -   d) 555 IAL (4 mmole) Et₃N    -   e) 231 mg (0.2 mmole) (Ph₃P)₄Pd    -   f) saturate with propyne gas with stirring at room temperature        (approx. 10 min.).

After 2 hours, more propyne gas is bubbled in and the reaction mixtureis stirred overnight at room temperature. The following morning morepropyne is bubbled in and stirred for an additional 2 hrs. To thereaction mixture is added Dowex ion-exchange resin (HCO₁-form), 10 mL ofMeOH and 10 mL of CH₂Cl₂ and stirring continued for 1 hr. The resin isfiltered off, washed with MeOH and the supernatant evaporated. SilicaGel chromatography yielded 517 mg (1.94 mmole, 97% yield) of product.See: Hobbs, J Org Chem (1989) 54: 3420-3422.

The purified material was protected with a 5′ DMT and phosphitylated asdescribed (Marugg, J. E., et al, Tetrahedron Letters (1986) 27:2661-2664) and used in solid phase synthesis as described (Froehler, B.C., et al, U.S. Pat. No. 4,959,463; Froehler, B. C., et al, TetrahedronLetters (1986) 27: 5575-5578).

The following notation is used to represent the bases in the designatednumbered oligomers in the Examples below: A, T, G, C, and U have theirconventional meanings. C′=5-methyl-2′-deoxycytidine, C*325-(1-propynyl)-2′-deoxycytidine; U*=5-(i-propynyl)-2′-deoxyuridine.

EXAMPLE 2 Formation of Triple Helix Structures Using Oligomers (ON)Containing 5-Propynyl Uracil Residues that Bind to Duplex DNA

Three oligomers were synthesized as follows:

-   -   ON-1 5′TC′TC′TC′TC′TC′TTTTT 3′ (SEQ ID NO: 1)    -   ON-2 5′TC′TC′TC′TC′TC′U*U*U*U*U* 3′ (SEQ ID NO:2)    -   ON-3 5′TC′TC′TC′U*C′U*C′U*TU*TU* 3′ (SEQ ID NO:3)

Each oligomer (ON) was hybridized with duplex DNA containing the targetsequence 5′ AGAGAGAGAGAAAAA 3′ (SEQ ID NO:4). Hybridization was carriedout in 140 mM KCl, 5 mM MgCl₂, 5 mM NaHPO₄, pH 6.6. Thermal stability,T_(m), of the resulting triple helix formed between each oligomer andthe target sequence was determined. The following T_(m) values wereobtained, ON—1 (control oligomer) was 42.1° C., ON-2 was 48.1° C. andON-3 was 55° C. The increased T_(m) values of ON-2 and ON-3 were notexpected and demonstrated that the triple helix formed was more stablethan the corresponding control triple helix structure.

EXAMPLE 3 Binding of Oligomers Containing 5-Propynyl Uracil or5-Propynyl Cytosine To Single-Stranded RNA

Oligomers were synthesized as follows:

-   -   ON-1 5′TC′TC′TC′TC′TC′TTTTT 3′ (SEQ ID NO: 1)    -   ON-3 5′TC′TC′TC′U*C′U*C′U*TU*TU* 3′ (SEQ ID NO:3)    -   ON-4 5′ TC*TC*TC*TC*TC*TTTTT 3′ (SEQ ID NO: 5)

The oligomers were hybridized with a single-stranded target RNAsequence, 5′ AAAAAGAGAGAGAGA 3′ (SEQ ID NO:6), in 140 mM KCl, 5 mMMgCl₂, 5 mM Na₂HPO₄, pH 6.6. The following T_(m) values for the duplexeswere obtained; ON—1 (control) was 65.5° C., ON-3 was 74.0° C. and ON-4was 73.0° C. Duplexes formed with ON-3 and ON-4 were more stable thanthe control oligomer. Surprisingly, ON-3 and ON-4 gave increased T_(m)values which demonstrated that the duplex formed was more stable thanthe corresponding control duplex structure.

EXAMPLE 4 Formation of Triple Helix Structures at Elevated pH

Triple helix formation at elevated pH was demonstrated using ON—1 as acontrol and ON-5,5′ U*C′U*C′U*C′U*C′U*C′U*U*U*U*U* 3′ (SEQ ID NO:7).Oligomers were hybridized with duplex target DNA, as described inExample 2 except that the buffer was at pH 7.4. T_(m) values of thetriple helix were then determined. ON-1 had a T_(m) of 27.1 while ON-5had a T_(m) of 51.5. Thus, oligomers containing 5-propynyl uracil werecapable of triplex formation at high pH levels, while the controloligomer formed triplex structure only at temperatures that are belowphysiological.

In an additional set of determinations, modified forms of ON-5 whereinthe 5-substituent of the deoxyuridine was, instead of propynyl,3-methylbutynyl (ON-5A) or 3,3-dimethyl butynyl (ON-5B), similar affectson the melting temperature of duplex and triple helices were observed.The results are shown in Table 1 below:

TABLE 1 Duplex^(a) Triple-helix^(a) @ RNA DNA pH = 6.60 T_(m) (° C.)T_(m) (° C.) T_(m) (° C.) ΔT_(m) (° C.) ON-1 63.0 54.5 39.6 — ON-5 79.065.5 64.8 +25.2 ON-5A 73.5 65.5 55.9 +16.3 ON-5B 68.5 66.0 42.5 +2.9^(a)T_(m) in 140 mM KCl/5 mM Na₂PO₄/1 mM MgCl₂, pH 6.60.

EXAMPLE 5 Synthesis of 5-(3-Methyl-L-Butynyl)-2′-DeoxyuridineH-Phosphonate, Oligomers Containing the Analog and Formation of TripleHelix Structures Using the Oligomers

5-(3-Methyl-1-butynyl)-2′-deoxyuridine H-phosphonate was synthesizedfrom 5-iododeoxyuridine essentially as described for5-(1-propynyl)-2′-deoxyuridine H-phosphonate in Example 1, except that 5equivalents of 3-methyl-1-butyne liquid was used in place of propyne.Silica gel purified material was then converted to the 5′-DMT,3′-H-phosphonate monomer and used in solid phase synthesis to generateoligomers as follows (ON-1 was used as a control containing thymine and5-methylcytosine as described in Example 2):

-   -   ON-1 5′TC′TC′TC′TC′TC′TTTTT 3′ (SEQ ID NO: 1)    -   ON-6 5′TC′TC′TC′U′C′U′CU′TU′TU′ 3′ (SEQ ID NO: 8)    -   ON-7 5′TC′TC′TC′TC′TC′U′U′U′U′U′ 3′ (SEQ ID NO:9)

Base residues designated U′ correspond to 5-(3-methyl-1-butynyl)uracil.The oligomers were hybridized with duplex DNA containing the targetsequence, 5′ AGAGAGAGAGAAAAA 3′ (SEQ ID NO:4).

EXAMPLE 6 Stability of Duplex and Triplex Structures and Inhibition ofTarget Gene Expression

Thermal stability. Additional oligomers were tested with respect tothermal denaturation or stability (T_(m)) after hybridization with DNAor RNA targets to form triplex or duplex structures respectively. TheDNA target used was an oligomer containing a self-complementary regionand having a 4 nucleotide loop at the end of the hairpin. The targetswere as follows:

DNA Duplex Target: 5′ AGAGAGAGAGAAAAAGGA ^(T)T(SEQ ID NO:10)

-   -   3′ TCTCTCTCTCTTTTTTTMCCT _(T)T (SEQ ID NO:11)

RNA Target: 5′ AAAAAGAGAGAGAGA 3′ (SEQ ID NO:12)

The assays for triple-helix binding were conducted in 140 mM KCL/5 mMNa₂HPO₄/5 mM MgCL₂ at pH=6.60 and the final concentration of alloligomers was −2 μM.

-   -   ON-1, set forth above, was used as a control.

Test oligomers 8-10 contain substitutions of 5-propynyluracil forthymine and 5-propynylcytosine for methylcytosine.

-   -   ON-8 5′ TC′TC′TC′U*C′U*C′U*TU*TU* 3′ (SEQ ID NO:3)    -   ON-9 5′ TC*TC*TC*TC*TC*TTTTT 3′ (SEQ ID NO: 5)    -   ON-10′ U*C* U*C* U*C* U*C* U*C*U*U*U*U*U* (SEQ ID NO: 54)

The results obtained showed that, with respect to triple-helixformation, the control ON-1 gave a T_(m) of 43.4° C.; ON-8 gave anelevated T. of 55.5° C.; and ON-9 gave a T_(m) of 26.3° C. ON-8containing U*, showed an increase in T_(m) of 2.4° C./substitution(ΔT_(m)@6.6=+12.1° C.) relative to ON-1 and ON-9, containing C*, showeda decrease in T_(m) of 3.4° C./substitution (ΔT_(m)@6.6=−17.1° C.)relative to ON-1. The T_(m) of a triple-helix, in which the third strandcontains 2′-deoxycytidines, is pH dependent, and the T_(m) at differentpH values (from 5.8 to 7.4) was used to examine the pH dependence of thecomplex. With all ON's the slope of the plot remained relativelyconstant (−18 to −20° C./pH unit).

The low T_(m) of ON-9, relative to ON's 1 and 8, can be explained interms of basicity of the heterocycle. Titration of the hydrocholoridesalt of C* and C′ showed that the pKa of the 5-propyne analog C* (3.30,±0.05) is 1.05 units less than the 5-methyl derivative C′ (4.35, ±0.05).The importance of protonation in triple-helix formation has beendemonstrated and the results above indicate that a decrease in basicityof the cytosine nucleobase has a dramatic effect on the stability of thecomplex. Surprisingly, the large difference in pKa's of the cytosines(C* and C′) has no significant effect on the slope of the T_(m) vs pHplot.

With respect to oligomer/RNA duplex formation, the control, ON-1 had aT_(m) of 65.5° C., ON-8 had a T_(m) of 74.0° C., ON-9 had a T_(m) of73.0° C. and ON-10 had a T_(m) of more than 90° C.; ON-8 containing U*,results in an increase in T_(m) of 1.7° C./substitution and ON-9,containing C*, results in an increase in T_(m) of 1.5° C./substitution.Under these conditions ON-10, containing complete substitution with U*and C*, has a T_(m) greater than 90° C. (approx. 1.7° C./substitution)indicating that the increases in binding affinity of these substitutionsare additive. These results show that the double-helix complex isgreatly stabilized by substitution with both C* and U* and, therefore,these analogs represent a new class of useful antisense ON's.

Binding assays were conducted using a combination of C* and U* inoligomers containing phosphorothioate internucleotide linkages as anadditional modification. Phosphorothioate linkages render oligomersnuclease stable, but reduce binding affinity for complementary targetsequences relative to unmodified phosphodiester linkages. Otherphosphodiester linkage analogs known in the art such asalkylphosphonate, methylphosphonate, phosphoroamidate and triesterlinkages suffer from similar limitations. Unexpectedly, oligomerscontaining a heterocycle modification that enhances binding affinity(defined herein as a positive modification), such as U* or C*, and amodification that reduces affinity (defined herein as a negativemodification), were found to have improved binding (i.e. a greaterbinding affinity than predicted by the additive effects—positive andnegative—of both modifications), relative to oligomers containing onlythe negative modification. Surprisingly, the propyne modificationcounteracts the negative binding effect of the phosphorothioate linkagesto an unexpected degree. That is, for oligomers containing T and C′ theΔT_(m) between phosphodiester and phosphorothioate is 14° C. (0.7° C.per substitution) while the ΔT_(m) with U* and C* is 6.0° C. (0.3° C.per substitution). These results clearly demonstrate a synergisticeffect between the negative modification (substitute linkage such asphosphorothioate) and the positive modification (base analog such as a5-substituted pyrimidine) wherein the positive modification compensatedto a degree that is more than additive with respect to binding affinity.

Binding results (ΔT_(m) relative to phosphodiester linkages) that wereobtained are shown in Table 2 below:

TABLE 2 ON Linkage ON Diester Thioate ΔT_(m)ATTTTC′TTC′ATTTTTTC′TTC′(SEQ ID NO:14) 54.0 40.0 −14.0AU*U*U*U*C′U*U*C′AU*U*U*U*U*U*C′U*U*C′ (SEQ ID NO:15) 76.5 68.5 −8.0AU*U*U*U*C*U*U*C*AU*U*U*U*U*U*C*U*U*C* (SEQ ID NO:16) 82.5 76.5 −6.0

Additional data obtained in vitro with respect to duplex formation withtarget RNA corresponding to T antigen (TAg) show that the binding of theoligomer to the target is sequence-specific for the 5-substitutedoligomers of the invention. The additional oligomers, 21 and 22, wereprepared; ON-22 is a scrambled form of ON-21 which is designed to targetthe T antigen coding region as described above.

-   -   ON-21: AU*U*U*U*C′U*U*C′AU*U*U*U*U*U*C′U*U*C′(SEQ ID NO: 17)    -   ON-22: U*U*AU*U*AU*C′U*U*C′U*U*C′U*U*U*U*C′U* (SEQ ID NO: 25)

The oligomers were tested in phosphodiester (ON-21, ON-22) andphosphorothioate (ON-21A, ON-22A) form; and ON-21B and ON-22B the2′-O-allyl T and C′ oligomers. The results are shown in Table 3:

TABLE 3 T_(m) ΔT_(m) ON-21 76.5 ON-22 53.0 23.5 ON-21A 68.0 ON-22A 42.026.0 ON-21B 70.0 ON-22B 45.0 25.0

The differences in T_(m)between the scrambled and unscrambled form areroughly the same regardless of the pyrimidine or linkage substitutionused.

Inhibition of Target Gene Expression

Additional oligomers designed to target T antigen (TAg) in amodification of the in vivo antisense assay described by Graessmann, M.et al. Nucleic Acids Res (1991) 19: 53-59 were also modified to containU* and/or C* as well as modified internucleoside linkages. The assayuses an expression vector for SV40 T antigen. Oligomers (ON-11 throughON-17) designed to target a sequence in the coding region were employed.The oligomers used in this assay were as follows:

-   ON 11: 5′ ATTTTC′TTC′ATTTTTTC′TTC′3′(SEQ ID NO:14)-   ON 12: phosphorothioate form of ON-11-   ON 13: 5′AU*U*U*U*C*U*U*C*AU*U*U*U*U*U*C*U*U*C* 3′(SEQ ID NO:16)-   ON 14: phosphorothioate form of ON-13-   ON 15: phosphorothioate 5′C*U*U*C*AU*U*U*U*U*U*C*U*U*C* 3′(SEQ ID    NO: 18)-   ON 16: phosphorothioate 5′C*U*U*C*AU*U*U*U*U*U*C*U* 3′ (SEQ ID NO:    19)-   ON 17: phosphorothioate 5′C*U*U*C*AU*U*U*U*U*U* 3′ (SEQ ID NO: 20)-   ON 18: phosphorothioate 5′C*U*U*C*AU*U*AU*U*U*C*U*U*C* 3′(SEQ ID NO:    21)-   ON 19: phosphorothioate 5′C*U*U*U*C*U*U*C*U*U*AC*U*U*C* 3′(SEQ ID    NO: 22)

ON 18 and 19 were mismatch controls and have the same base compositionfound in ON 15 with sequence mismatches as shown in bold. The T_(m) ofthe oligomers with the complementary RNA were determined as describedabove. The nuclease stability of the oligomer in the cells and theability of the oligomer to inhibit T antigen synthesis was alsodetermined. The ability of ON 11 to ON 17 to confer RNase H sensitivityon the bound RNA was also determined and each oligomer was found toconfer RNase H sensitivity. Details of the antisense assay protocol aredescribed in Example 7. The results are shown in Table 4.

TABLE 4 TAg Oligomer RNA T_(m)* S.N.** IC₅₀*** ON-11 54.0° C. − − ON-1240.0° + n.s. ON-13 82.5° − 2.5 ON-14 76.5° + 0.05 ON-15 71.0° + 0.10ON-16 63.5° + 0.25 ON-17 53.5° + 1.0 ON-18 59.5° + 0.50 ON-19 43.0° + −*T_(m), thermal stability of duplex determined under the same conditionsas previously described at pH 6.6 in 1 mM MgCl₂. **S.N., stability tonuclease digestion in live cells at 37° C.; (−) nuclease sensitive, (+)nuclease resistant. ***IC₅₀, oligomer concentration (μM) showing 50%inhibition of TAg expression; (−), no inhibition of TAg expressiondetected; (n.s.), nonspecific inhibition at 25 μM.

As seen, substituting the phosphorothioate linkage for phosphodiesterdecreases the affinity of the oligomer for target RNA but enhances thenuclease stability of the oligomers in the cell. Replacement of thethymine and cytosine bases by the 5-substituted bases of the inventionenhanced affinity of the oligomers. At an increased concentration ofoligomer (diester, ON-13), the enhanced affinity of the oligomer led todetectable T antigen synthesis inhibition. The phosphorothioate analogcontaining the modified bases is sufficiently stable and has sufficientaffinity for the target RNA to effect inhibition of the synthesis of Tantigen. The increasing IC₅₀ value coupled with the decreasing T_(m) ofON 18 and ON 19 relative to ON-15 indicated that these oligomers werebinding to target sequences less effectively as the number of mismatchesincreased. These results demonstrate sequence-specific inhibition oftarget gene expression by invention antisense oligomers.

In addition to inhibition of TAg synthesis, a phosphorothioate oligomer,ON 20, 5′ U*U*GC′C′GU*U*U*U*C′AU*C′AU*AU*U*U*AAU* 3′ (SEQ ID NO:23),that is complementary to the β-galactosidase RNA, was able to inhibitβ-galactosidase in a sequence specific manner with an IC₅₀ of 0.25 μm.ON 20A, ON 20 with T and C′, did not inhibit β-galactosidase expressionin a sequence specific manner.

EXAMPLE 7 Assay Method and Inhibition of Target Gene Expression

Assay Method. Antisense oligomers were evaluated for biological efficacyin vivo using a microinjection procedure. The protocol differs frompreviously described procedures by utilizing an additional coinjectedgene which serves as an internal control for transfected gene expression(Graessman, M., et al., Nucleic Acids Res (1991) 19: 53-59).Microinjections were performed using 5-10 copies per cell of pSV40plasmid expressing the TAg gene along with varying amounts of antisenseoligomers targeted to different regions to the TAg RNA. Coinjectionmarkers such as 40 copies of plasmid per cell containing theβ-galactosidase gene under the control of the RSV promoter or thechloramphenicol acetyl transferase gene under the control of the SV40promoter were used. Marker genes are those which generate proteins thatcan readily be measured so that specificity of gene expressioninhibition can be shown. The antisense oligomer does not affect theability of the cells to continue to produce protein products of themarker gene. Suitable marker genes generate chloramphenicolacetyltransferase (CAT), β-galactosidase, luciferase or cell surfaceantigens, enzymes or markers for which assay methods are known. Incontrol experiments without antisense oligomer, 100% of microinjectedcells expressed the β-galactosidase protein at 4.5 h after injectionwhile approximately 60% of microinjected cells expressed the TAgprotein, as detected by dual label immunofluorescent labelingtechniques. Target sequences in the TAg RNA included a coding sequenceapproximately 150 bases from the translation initiation AUG codon,sequences in the 5′-untranslated region and sequences at the AUG codon.Antisense oligomers from 9 to 20 bases in length were examined usingconcentrations of oligomers of between 5 nM and 25 μM and the compoundswere assayed at times ranging from 4.5 to 72 hours postinjection. CV1 orRat2 cells were microinjected using conditions essentially as described(Graessman, M., et al., ibid.).

Target Sequence Binding and Target Gene Inhibition. An oligomer (5′ATTTTC′TTC′ATTTTTTC′TTC′ 3′(SEQ ID NO:14)) was systematically varied,using the phosphodiester antisense oligomer, ON-11, as a control. Thephosphorothioate analog, ON-12, of the same oligomer was also prepared,but had no altered bases. The corresponding oligomer having the5-substituted bases of the invention universally substituted wasprepared both in the phosphodiester, ON-13, and phosphorothioate form,ON-14; finally, the 2′-O-allyl-substituted form was tested as well.

As shown in Table 4, substituting the phosphorothioate linkage forphosphodiester decreases the affinity for target RNA but enhanced thenuclease stability. Analysis of the time course of inhibition of Tantigen expression showed that ON-13 (phosphodiester linked oligomercontaining U* and C*) had activity that was detectable until 6 hoursafter microinjection into cells at a concentration of 25 μM. Bycontrast, ON-14 (phosphorothioate linked oligomer containing U* and C*with the same sequence as ON-13) was active for 48 hours aftermicroinjection of 0.5 μM oligomer into cells.

Both the 9-mer (5′C*U*U*C*AU*U*U*U* 3′ (SEQ ID NO:24)) and 11-mer(ON-17) phosphorothioates were able to inhibit T antigen synthesis whenthey contain the 5-substituted bases of the invention. However, the9-mer had relatively weak sequence-specific inhibitory activity.

Also tested in the foregoing assay were an oligomer (5′ UT) designed tobind to the 5′ untranslated region of the T antigen near the CAP site ofthe mRNA and an oligomer with a sequence designed to bind to the regionof the start codon. These oligomers have the sequences shown:

-   -   5′ UT oligomer: 5′-GCC TCC TCA CTA CTT CTG GA-3′(SEQ ID NO:26)    -   AUG oligomer: 5′-CAT CTT TGC AAA GCT TTT TG-3′(SEQ ID NO:27)

The phosphorothioate form of the 5′UT and AUG oligomers composed ofthymine and 5-methylcytosine bases was unable to effect detectableinhibition at 20 μM. However, the phosphorothioate analogs wherein5-propynyluracil was substituted systematically for thymine residuesshowed 100% inhibition at 1 μM.

Further experiments with this assay system using a T antigen targetsequence in the 5′ untranslated region demonstrated that substitution ofthe modified oligomers of the invention containing phosphodiesterlinkages but containing 2′-O-allyl substitutions in the oligomerscontaining fully substituted nucleomonomers wherein C* replaces C and U*replaces T are capable of inhibiting T antigen synthesis. Table 5 showsthe results obtained using the 5′UT oligomer. Oligomers 1-4 were 20-mershaving the sequence shown above. Oligomer 5 had the underlined sequenceshown above and 5-propynyluridine substituted for thymidine and5-propynylcytidine substituted for cytidine.

TABLE 5 Oligomer* Tm IC50 (μM) 1. 2′-O—Me (U, C) — n.i.** 2. Thioate (T,C′) 70.5 2.5 3. Thioate (U^(•), C′) 81.0 0.5 4. Thioate (U^(•),C^(•)) >90.0  0.25 5. 2′-O-allyl (U^(•), C^(•)) >90.0 5.0 *Thioate orphosphorothioate linkages **No detectable inhibition at 5 μM

As shown in Table 5 various combinations of inclusion of U* and C* alongwith either a phosphorothioate backbone or a 2′-O-allyl substitutionprovided inhibition. Although oligomer 5 is not a substrate for RNase H,inhibition of TAg expression was observed. Inhibition mediated byoligomer 5 is believed to result from the high affinity and nucleasestability that the 2′-O-allyl modification confers. Incorporation of U*and/or C* into oligomers containing full or partial 2′-O-allylmodification will provide oligomers that can be used to inhibit targetgene expression.

In addition to sequences in the 5′UT, AUG codon region and exondescribed above, TAg sequences at TAg intron/exon junction, exon/exonjunction and in an intron were targeted using 15-mer phosphorothioateoligomers that were fully substituted with U* and/or C* according to thetarget sequences. The oligomers contained from about 50% to about 70% ofU* and/or C* bases in the oligomer. All of the oligomers effectivelyinhibited TAg synthesis. These results indicated that the high affinityoligomers were capable of inhibiting gene expression at locationsthroughout the RNA and were thus effective in spite of any secondarystructure that can have been present in the TAg RNA.

EXAMPLE 8 Delivery of Oligomers into Cells

ON-15 was tested for inhibition of T antigen (TAg) expression using themethod described in Example 7. ON-15 was incubated for 24 hours with CV1cells at an extracellular concentration of 50 μM in tissue culturemedium, followed by microinjection of TAg and 8-galactosidase expressionplasmids. 4.5 hours after injection, cells were fixed and stained forTAg expression. By comparison with microinjected ON-15, whichefficiently inhibits TAg expression, ON-15 incubated in extracellularmedium was much less efficient with no detectable inhibition of TAgsynthesis. The experiment was repeated using ON-15 derivatized at the 5′end with fluorescein-aminohexanol (Fl-ON-15) at an extracellularconcentration of 50 μM which was incubated with cells for 24 hours.Control cells were microinjected with Fl-ON-15 at an intracellularconcentration of 0.5 μM along with TAg and β-galactosidase expressionplasmids. Microinjected Fl-ON-15 was localized in the nucleus whileFl-ON-15 added to the extracellular medium was localized in cytoplasmiccompartments that resembled endosomes and lysosomes. The same pattern ofresults were obtained in CV1, Rat2, HeLa, SKOV-3, BUD-8, BC3H1 andccd45sk cell lines. Oligomers of the invention are active in differentmammalian species and are thus suitable for modulating gene expressionregardless of the species.

Fl-ON-15 and ON-15 were delivered to cellular cytoplasm using acommercially available cationic lipid preparation, Lipofectin™(BRL-Gibco, cat. no. 8292SA). A Lipofectin™ concentration of 10 μM inOptimem (BRL-Gibco) was used according to manufacturer's instructions.DOTMA is the lipid present in Lipofectin™. Cells were incubated inOptimem containing either the Fl-ON-15-lipid or ON-15-lipid preparationfor 4 hours, followed by incubation of the cells for 16 hours instandard medium (DMEM with 10% FBS for CV1 cells). Immunofluorescenceanalysis of treated cells showed that about 90% of the cells containedFl-ON-15 localized in the nucleus. Delivery of ON-15 to cells wasassayed by microinjection of TAg and 8-galactosidase expression plasmidsinto cells after incubation with the lipid-oligomer complex. ON-15inhibited TAg expression with an IC₅₀ of less than 5 nM usingLipofectin™ at a 10 μM concentration. Preparations of oligomer complexedwith cationic lipid (such as Lipofectin™) were thus capable ofdelivering oligomers containing a label such as fluorescein and/or5-modified pyrimidines such as U* or C* to the cellular cytoplasm,indicating that modifications incorporated into oligomers, such as baseanalogs or a label, do not interfere with formation cationiclipid-oligomer complexes.

In an alternative protocol, Fl-ON-15 was also delivered into cells by atransfection protocol using DMSO. CV1 cells were incubated in DMEM-10%FBS medium containing 1% DMSO and 1 μM Fl-ON-15 for 4 hours at 37° C.Fluorescence microscopy demonstrated that the oligomer was localized inthe nucleus of about 20% of the treated cells.

Fl-ON-15 was synthesized by coupling a commercially availableaminohexane amidite (Glen Research) to the 5′-OH of ON-15 using standardcoupling conditions. The free amine was then linked to fluorescein-NHSester (Molecular Probes) to generate Fl-ON-15. Synthesis offluorescein-linked oligomer can also be accomplished using fluoresceinamidite or fluorescein-CPG according to manufacturer's instructions(Glen Research).

EXAMPLE 9 Synthesis of 2′-O-Allyl Monomers for Oligomer Synthesis

Preparation of 5-propynyl-2′-O-allyluridine nucleomonomer. 343 mg (0.50mmole) of 14 (FIG. 4) was dissolved into anhydrous CH₃CN (5 mL) and tothis was added 2-pyridinealdoxime (67 mg, 0.55 mmole) and1,1,3,3-tetramethylguanidine (75 μL, 0.6 mmole) at room temperature.After 18 hr the reaction mixture was diluted with EtOAc and washed withaq. citric acid (0.1 M). The aqueous layer was extracted with EtOAc, thecombined organic layers washed with saturated aq. NaHCO₃ (3 times),dried over Na₂SO₄ and evaporated. The residue was dissolved into EtOAc(5 mL) and to this was added 1 M TBAF/THF (1.5 mL, 1.5 mmole), thesolution stirred for 1 hr and diluted with EtOAc. The solution waswashed with saturated aq. NaHCO₃ (2 times), the combined aqueous layerextracted with EtOAc (3 times), the combined organic phase dried overNa₂SO₄ and evaporated. The residue was evaporated from anhydrouspyridine (10 mL), dissolved into anhydrous pyridine (5 mL), and to thiswas added dimethoxytrityl chloride (200 mg, 0.6 mmole) and the solutionstirred for 18 hr. The reaction mixture was evaporated to approximately2 mL, diluted with CH₂Cl₂, washed with saturated aq. NaHCO₃, dried overNa₂SO₄ and evaporated. Purification by silica gel chromatography(EtOAc/Hexane, 1/1) yielded 197 mg (0.32 mmole, 64%) of 6 shown in FIG.4.

Preparation of 5-propynyl-2′-O-allylcytidine nucleomonomer. 343 mg (0.50mmole) of 14 was dissolved into anhydrous CH₃CN (10 mL), and thesolution transferred to a Parr Bomb, cooled to 0° C., and saturated withNH₃. This was placed in an 80° C. bath for 24 hr (75 psi), cooled toroom temperature and evaporated to dryness. The residue was evaporatedfrom anhydrous DMF (10 mL), dissolved into anhydrous DMF (5 mL), and tothis was added diisobutylformamide dimethylacetal (0.2 mL, 0.84 mmole)at room temperature. After 18 hr H₂O (25 μL) was added, the solutionevaporated, dissolved into EtOAc (5 mL) and to this was added 1 MTBAF/THF (1.5 mL, 1.5 mmole). After 1 hr the reaction mixture wasdiluted with EtOAc, washed with saturated aq. NaHCO₃, dried over Na₂SO₄and evaporated. The residue was evaporated from anhydrous pyridine (10mL), dissolved into anhydrous pyridine (5 mL), and to this was addeddimethoxytrityl chloride (200 mg, 0.6 mmole) and the solution stirredfor 5 hr. The reaction mixture was evaporated to approximately 2 mL,diluted with CH₂Cl₂, washed with saturated aq. NaHCO₃, dried over Na₂SO₄and evaporated. Purification by silica gel chromatography (EtOAC/Hexane,from 2/3 to 3/2) yielded 242 mg (0.32 mmole, 641) of 8 shown in FIG. 4.

EXAMPLE 10 Formacetal Dimer Synthesis

FIG. 11 shows a synthesis scheme that was used to obtain a formacetallinked 5-propynyl-2′-deoxyuracil dimer. The synthesis protocolintroduced the propynyl substituent at the level of a dimer byconversion of the 5-iodouracil precursor as shown. A similar protocolcan be used to convert trimers, tetramers, pentamers or longer 5-iodoprecursors to the 5-propynyl product in a similar fashion. Thissynthetic method gave unexpectedly high yields of the 5-propynylproduct.

EXAMPLE 11 3′-Thioformacetal Dimer Synthesis

Preparation of (26): (25) was suspended into CH₂Cl₂ and paraformaldehyde(1.6 eq) was added, the suspension cooled to 0° C. and HCl (anhydrous)passed through the solution for about 10 minutes. The suspension wassealed and stored at 0-5° C. for 4 hours. The resulting solution wasdried, filtered, and evaporated to yield (26).

Preparation of 10:3′-Deoxy-3′-thioacetyl-5′-dimethoxytrityl-5-propynyl-2′-deoxyuridine wasdissolved in methanolic ammonia in a flask that had been flushed with O₂and the solution was sealed and stirred for 1 hour. The solvent wasremoved and the residue dissolved in EtOAc and washed with NaHCO₃ andbrine. The organic phase was dried, evaporated and purified by silicagel chromatography (MeOH/CH₂Cl₂). The resulting disulfide was dissolvedinto dioxane/H₂O followed by addition of tributylphosphine (1.0 eq) andthe solution stirred for 30 minutes. The solvent was removed and crudecompound 10 was used directly to prepare (27).

Preparation of (27): Compound 10 (1.1 eq) was dissolved in DMF and DIPEA(diisopropylethylamine, 2.5 eq) was added and the solution was cooled to0° C. under Ar. A solution of compound (26) (1 eq, in DMF) was added,the solution stirred for 10 hours, diluted, extracted against H₂O,dried, evaporated and purified by silica gel chromatography(MeOH/CH₂Cl₂).

Preparation of 11: (27) was dissolved in methanolic ammonia and stirredin a sealed flask at room temperature for 3 hours. The solvent wasremoved and purified (Silica Gel, MeOH/CH₂Cl₂ (0-4% MeOH)) and thepropyne moiety introduced has described in Example 1.

Preparation of 11A: 11 was phosphitylated using standard procedures.

EXAMPLE 12 5-(2-Pyridinyl)-2′-Deoxyuridine and5-(2-Pyridinyl)-2′-Deoxycytidine Synthesis

2-Trimethylstannylpyridine. A solution of 15.7 mL of 1.6 Mn-butyllithium (25.1 mmol) in anhydrous ether (25 mL) was stirred underAr at −78° C. and to this was added a solution of 2-bromopyridine (3.97g, 25.1 mmol) in anhydrous ether (12.5 mL). The resulting orangesolution was stirred for 2 h, and trimethyltin chloride in THF (26.0mmol, 26 and, 1.0 M) added over 30 min. The reaction was stirred for 1 hat −78° C. then warmed to room temperature over 1 h, filtered and thefiltrate evaporated. Distillation afforded 3.1 g (51%) of the titlecompound as a colorless liquid which solidified in the receiver flask.B.p. 65° C./2 mm Hg; Literature B.p. 75° C./4 mm Hg.

5-(2-pyridinyl)-2′-deoxyuridine. In a 25 mL pear shaped flask was placed5-iodo-2′-deoxyuridine (0.354 g, 1.0 mmol), 2-trimethylstannylpyridine(0.84 g, 3.5 mmol), Bis (triphenylphosphine)-palladium (II) chloride(0.070 g, 0.1 mmol), and anhydrous 1,4-dioxane (15 mL). The reaction washeated at 60° C. for 15 hrs then at 90° C. for 1 h. The solvent wasevaporated and the residue purified by silica gel chromatography (10%CH₃OH in CH₂Cl₂ (1% NH₃)) to yield 0.253 g (83%) of the title compoundas a white solid m.p. 201-203° C.

5-(2-Pyridinyl)-2′-deoxycytidine. In a 25 mL pear shaped flask wasplaced 5-iodo-2′-deoxycytidine (0.425 g, 1.2 mmol),2-trimethylstannylpyridine (1.4 g, 5.8 mmol), Bis(triphenylphosphine)-palladium (II) chloride (0.084 g, 0.12 mmol), andanhydrous 1,4-dioxane (15 mL). The reaction was heated at 60° C. for 15hrs then at 90° C. for 1 h. The solvent was evaporated and the residuepurified by silica gel chromatography (10% CH₃OH in CH₂Cl₂ (1% NH₃)) toyield 0.173 g (47%) of the title compound as a white solid m.p. 197-198°C.

EXAMPLE 13

Synthesis of 5-(Thienyl)-5′-Dimethoxytrityl-2′Deoxyuridine2-Trimethylstannylthiophene. To a solution of thiophene (2.1 g, 25.0mmol) in anhydrous THF (50 mL) was added dropwise over 30 minutes at −38C t-butyllithium in pentane (1.6 M, 16.0 mL, 25.6 mmol). The solutionwas stirred for 1 h at −38 C and then cooled to −78 C. Trimethyltinchloride in THF (1 M, 25.3 mL, 25.3 mmol) was added dropwise over 30min. The reaction was then stirred for 2 h at −78 C and then for 15 h atr.t. The solvent was evaporated and the resulting yellow solid was takenup in ether, washed with water and dried Na₂SO₄ and then evaporated togive a light brown liquid which solidified on standing.

5-(2-thienyl)-5′-dimethoxytrityl-2′-deoxyuridine. In a 50 mL flask wasplaced 5-iodo-dimethoxytrityl-3′-O-toluolyl-2′-deoxyuridine (2.15 g,2.77 mmol), 2-trimethylstannylthiophene (1.85 g, 7.5 mmol),Bis(triphenylphosphine)-palladium (II) chloride (0.196 g, 0.28 mmol) andanhydrous THF (30.0 mL). The was heated at 73 C for 18 h, then cooled toroom temperature. The resulting black precipitate was filtered andwashed with THF. The solvent was evaporated and the green residue wasdissolved in ethyl acetate (100 mL), washed with water (50 mL) and dried(Na₂SO₄). The solvent was evaporated and the residue was taken up indioxane/conc. NH₄OH (1/1, 80 ml), stirred for 18 h at r.t. andevaporated. Silica gel purification yielded 0.430 g (25%) of5-(2-thienyl)-5′-dimethoxytrityl-2′-deoxyuridine.

EXAMPLE 14 Synthesis of 5-((1-Ethynyl)-2-Pyrimidinyl)-2′-Deoxyuridine

2-Iodopyrimidine. Liquid HI (28.0 mL, 212 mmol) was stirred vigorouslyat 0° C. and solid 2-chloropyrimidine (7.0 g, 61 mmol) was added slowlyso that the temperature did not rise above 8° C. The reaction wasstirred at 5° C. for 1 h., solid potassium carbonate (14.7g, 106 mmol)was arefully added over 0.5 h. (temperature less than 10c), and thereaction was decolorized by addition of a small amount of solid sodiumbisulfite. The solution was extracted with eiher (5×50 mL), dried(Na₂SO₄), evaporated and the residue was recrystalized from hexane toafford 7.11 g (56%) of the title compound, m.p. 29-30° C.2-((Trimethylsilyl)ethynyl)pyrimidine. In a 50 mL flask was placed2-iodopyrimidine (20 .g, 9.7 mmol), copper (I) iodide (9.5 mg, 0.05mmol), triethylamine (0.7 mL, 5.0 mmol),Bis(triphenylphosphine)-palladium (II) chloride (0.07 g, 0.1 mmol) andanhydrous dioxane (10 mL). The reaction was heated at 60° C. for 18hours then at 95° C. for 8 h. The solvent was evaporated and the residuewas dissolved in CH₂Cl₂ (200 mL), washed with H₂O (40 mL), dried(Na₂SO₄) and evaporated. The product was purified by silica gelchromatography (CH₂Cl₂) to yield 1.06 g (60%) of the title compound.

2-ethynylpyrimidine. To a solution of2-((Trimethylsilyl)ethynyl)pyrimidine (1.06 g, 6.0 mmol) in anhydrousmethanol (9 mL) was added solid potassium carbonate (0.083 g, 0.6 mmol)and the reaction stirred for 2.5 h. The solid was removed by filtration,washed with methanol (10 mL) and the supernatant evaporated. The residuewas dissolved in CH₂Cl₂ (30 mL), washed with saturated NaHCO₃, dried(Na₂SO₄) and evaporated. Silica gel chromatography (1-2% CH₃OH inCH₂Cl₂) yielded 0.400 g (64%) of the title compound. This was coupled to5-Iodo-2′-deoxyuridine and converted to the protected H-phosphonate asin Example 1.

EXAMPLE 15 Binding of Oligomers containing U* and 3′-Thioformacetal orFormacetal Linkages

The following oligomers were synthesized. Unless otherwise indicated,all linkages were phosphodiester. The symbol, *, indicates a3′-thioformacetal linkage (3′,5′), and the symbol, O, indicates aformacetal linkage (3′,5′).

-   ON-1 5′TC′TC′TC′TC′TC′TTTTT 3′ (SEQ ID NO:1)-   ON-23 5′TC′TC′TC′TC′TC′U*U*U*U*T 3′ (SEQ ID NO:28)-   ON-24 5′TC′TC′TC′TC′TC′T.TT.TT 3′ (SEQ ID NO:29)-   ON-25 5′TC′TC′TC′TC′TC′U*.U*U*.U*T 3′ (SEQ ID NO:30)-   ON-26 5′TC′TC′TC′TC′TC′U*oU*U*oU*T 3′ (SEQ ID NO:31)

The oligomers were tested with respect to thermal denaturation (T_(m))or stability after hybridization with DNA or RNA targets to form triplexor duplex structures respectively. The DNA target used was an oligomercontaining a self-complementary region and having a 4 nucleotide loop atthe end of the hairpin as shown. The targets were as follows:

DNA Duplex Target: 5′ AGAGAGAGAGAAAAAGGA ^(T)T(SEQ ID NO:10)

-   -   3′TCTCTCTCTCTTTTTCCT _(γ) T (SEQ ID NO: 11)    -   RNA Target: 5′ AAAAAGAGAGAGAGA 3′ (SEQ ID NO:12)

The assays for binding were conducted in 140 mM KCL/5 mM Na₂HPO₄/1 mMMgCL₂ at pH=7.2 for single stranded RNA or DNA (duplex hybridization)and at pH 6.6 for duplex DNA (triplex hybridization conditions). Thefinal concentration of all oligomers was ˜2 μM. T_(m) data obtained withthe oligomers is shown in Table 6.

TABLE 6 Duplex T_(m) Triplex ON RNA DNA T_(m) ON-1 62.5 55.5 38.9 ON-2369.0 59.5 45.0 ON-24 62.0 53.0 41.5 ON-25 71.5 61.5 47.6 ON-26 69.0 59.545.5

EXAMPLE 16 Binding of Oligomers Containing5-(2-Pyridinyl)-2′-Deoxyuridine (U^(P)) or5-(2-Pyridinyl)-2′-Deoxycytidine (C^(P)) and5-(2-Thienyl)-2′-Deoxyuridine (U^(T))

The following oligomers were synthesized. All linkages werephosphodiester

-   ON-1 5′TC′TC′TC′TC′TC′TTTTT 3′ (SEQ ID NO:1)-   ON-28 5′TC′TC′TC′TC′TC′U^(P)U^(P)U^(P)U^(P)U^(P) 3′ (SEQ ID NO:32)-   ON-29 5′TC′TC′TC′U^(P)C′U^(P)C′U^(P)TU^(P)TU^(P) 3′ (SEQ ID NO:33)-   ON-30 5′TC^(P)TC^(P)TC^(P)TC^(P)TC^(P)TTTTT3′ (SEQ ID NO:34)-   ON-43 5′TC′TC′TC′TC′TC′U^(T)U^(T)U^(T)U^(T)U^(T)3′ (SEQ ID NO:35)-   ON-44 5′TC′TC′TC′U^(T)C′U^(T)C′U^(T)TU^(T)TU^(T) 3′(SEQ ID NO:36)

The oligomers were tested with respect to thermal denaturation (T_(m))or stability after hybridization with ssDNA or ssRNA targets to form aduplex and with dsDNA to form a triplex. The DNA target used was anoligomer containing a self-complementary region and having a 4nucleotide loop at the end of the hairpin as shown. The targets were asfollows:

DNA Duplex Target: 5′ AGAGAGAGAGAAAAAGGA ^(T)T (SEQ ID NO: 10)

-   -   3′TCTCTCTCTCTTTTTCCT _(γ)T (SEQ ID NO:11)

DNA/RNA Target Sequence: 5′AAAAAGAGAGAGAGA 3′ (SEQ ID NO:12)

The assays for triple-helix binding were conducted in 140 mM KCL/5 mMNa₂HPO₄/1 mM MgCL₂ at pH=7.2 for single stranded RNA or DNA (duplexhybridization) and at pH=6.6 for duplex DNA (triplex hybridizationconditions). The final concentration of all oligomers was ˜2 μM. T_(m)data obtained with the oligomers is shown in Table 7.

TABLE 7 Duplex T_(m) Triplex ON RNA DNA T_(m) ON-1 62.5 55.0 39.6 ON-2864.0 54.0 41.8 ON-29 62.5 52.0 31.7 ON-30 61.5 46.5 — ON-43 64.5 55.031.6 ON-44 63.5 52.5 19.9

EXAMPLE 17 Binding of Oligomers Containing5-(1-Propynyl)-2′-Deoxyuridine (U*) and carbocyclic 5-Methyl-2′Deoxycytidine(C#) or 8-Oxo-N⁶-Methyl-2′-Deoxyadenosine (M)

The following oligomers were synthesized. All linkages werephosphodiester.

-   ON-1 5′TC′TC′TC′TC′TC′TTTTT 3′ (SEQ ID NO:1)-   ON-32 5′U*C#U*C#U*C#U*C#U*C#U*U*U*U*U* 3′ (SEQ ID NO:37)-   ON-33 5′TTTMTTTMMTMMTTTTT 3′ (SEQ ID NO: 38)-   ON-34 5′U*U*U*MU*U*U*MMU*MMU*U*U*U*U* 3′ (SEQ ID NO: 39)

The oligomers were tested with respect to target sequence binding byfootprint analysis using DNase digestion after binding with duplex DNAto form triplex structures. Hybridization was conducted at 37 C forabout 1 hour in 140 mM KCL, 10 mM NaCl, 1 mM MgCl, 1 mM sperminehydrochloride, MOPS pH 7.2 using target DNA at approximately 1 nM. Thetarget sequence was as follows:

DNA Duplex Target: 5′ AGAGAGAGAGAAAAA 3′ (SEQ ID NO:4)

-   -   3′ TCTCTCTCTCTTTTT 5′ (SEQ ID NO:40)

The sequence was contained on a gel purified 370-bp restriction fragmentderived from a cloning vector. The concentration of ON-1 and ON-32 wasvaried from 0.01 to 10.0 μM. The nuclease protection results obtainedwith ON-1 and ON-32 indicated that ON-32 had a binding affinity forduplex DNA that was about 1000-fold greater than the affinity of ON-1for the same target.

The ON-33 and ON-34 target sequence was as follows:

DNA Duplex Target: 5′ AAAGAAAGGAGGAAAAA 3′ (SEQ ID NO:41)

-   -   3′ TTTCTTTCCTCCTTTTT 5′ (SEQ ID NO:42)

The sequence was contained in a plasmid that was linearized byrestriction enzyme digestion. The sequence is found in the promoterregion of the IL-1 gene. The assays for triple-helix binding wereconducted in 140 mM KCL/10 mM NaCl/1 mM MgCL₂/1 mM sperminehydrochloride/20 mM MOPS pH 7.2. The final concentration of the targetwas −2 nM while the concentration of ON-33 and ON-34 was varied from 0.1to 10.0 μM. The nuclease protection results obtained with ON-33, whichgave full DNase protection at 0.1 μM, and ON-34, which gave full DNaseprotection at a concentration well below 0.1 μM, indicated that ON-34had a binding affinity for duplex DNA that was greater than or equal to10-fold greater than the affinity of ON-33 for the same target.

The results obtained with these oligomers indicate that the inventionbases are compatible with other triplex binding competent base analogsand can thus be used to design high affinity oligomers withtriplex-competent bases and base analogs as desired.

EXAMPLE 18 Duplex Formation Using Oligomers Containing5-(1-Propynyl-2′-O-Allyluridine (U^(X)) or5-(1-Propynyl)-2′-O-Allylcytidine (C^(X))

The following oligomers were synthesized. All linkages werephosphodiester. Nucleomonomers were prepared as described in Example 9.5- Methyl-2′-O-allyluridine, T′, and 5-methyl-2′-O-allylcytidine, C″,were used in addition to U^(X) and U^(X).

-   ON-1 5′TC′TC′TC′TC′TC′TTTTT 3′ (SEQ ID NO: 1)-   ON-35 5′TC′TC′TC′TC′TC′T′T′T′T′T′3′ (SEQ ID NO:43)-   ON-36 5′TC′TC′TC′TC′TC′U*U*U*U*U* 3′ (SEQ ID NO:44)-   ON-37 5′TC′TC′TC′TC′TC′U^(X)U^(X)U^(X)U^(X)U^(X)3′ (SEQ ID NO:45)-   ON-38 5′TC″TC″TC″TC″TC″TTTTT 3′ (SEQ ID NO:46)-   ON-39 5′TC*TC*TC*TC*TC*TTTTT3′ (SEQ ID NO:47)-   ON-40 5′TC^(X)TC^(X)TC^(X)TC^(X)TC^(X)TTTTT 3′(SEQ ID NO:48)

The oligomers were tested with respect to thermal denaturation (T_(m))or stability after hybridization with an RNA target to form duplexstructures. The target was as follows:

RNA Target: 5′ AAAAAGAGAGAGAGA 3′ (SEQ ID NO: 49)

The assays for duplex-helix binding was conducted in 140 mM KCL/5 mMNa₂HPO₄/1 mM MgCL₂pH=6.6 for the RNA target. The final concentration ofall oligomers was −2 μM. T_(m) data obtained with the oligomers is shownin Table 8.

TABLE 8 ΔT_(m) ON T_(m) (° C./substitution) ON-1 63.0 — ON-35 64.5 +0.3ON-36 70.5 +1.5 ON-37 71.5 +1.7 ON-38 66.5 +0.7 ON-39 70.0 +1.4 ON-4073.0 +2.0

The results obtained from these analyses demonstrated that the inventionbases can be combined with modified sugars in binding competentoligomers. Enhanced binding due to the 2′-O-allyl sugar modification andto the invention bases give significantly increased binding affinitycompared to either modification alone. The 2′-O-allyl modificationrenders oligomers incompetent as a substrate for RNase H. Thus, RNAsbound by oligomers containing 2′ modifications and the invention basescan be advantageously used as probes for RNA sequences in cellularextracts containing nucleases.

EXAMPLE 19 Triplex Formation Using Oligomers Containing U* and aSwitchback Linker (X*) Containing U*

The following oligomers were synthesized. All linkages werephosphodiester.

-   ON-41 5′U*C′U*C′U*U*U*U*U*U*C′U*U*C′U*C′U*U*U*C′X*U*U*U*U*U*U*U* 3′    (SEQ ID NO:50)-   ON-42 5′U*C′U*C′U*U*U*U*U*U*C′U*U*C′U*C′U*U*U*C′X*U*U 3′ (SEQ ID    NO:51)

The DNA target sequence used was:

DNA Duplex Target: 5′ AGAGAAGGGAGAAGAGAAAGAAATTTTTTTTT 3′(SEQ ID NO:52)

-   -   3′TCTCTTTTTTCTTCTCTCTTTCTTTAAAAAAAAA 5′(SEQ ID NO:53)

The target sequence was introduced into an intron of the TAg gene andwas used in DNase protection analysis as a linearized restrictionfragment at about 2 nM. The switchback linker synthon, X*, had thestructure 9 shown in FIGS. 5-1 and was incorporated into ON-41 and ON-42by standard H-phosphonate chemistry. The switchback linker wasassociated with 2 null base pairs (bases in the target that were nothybridized with). Full DNase protection of the target sequence wasobtained using either ON-41 or ON-42 at a concentration less than 100nM.

These results demonstrate that the invention bases are compatible withswitchback linkers, such as the o-xyloso linker described here.

EXAMPLE 20 Triple Helix Formation Using Oligomers Containing U* andPhosphorothioate Linkage

Triple helix formation was demonstrated using ON-5 and thephosphorothioate form of ON-5 as described in Example 16. The resultsshow that ON-5 has a T_(m) of 55.8° C. and the phosphorothioatederivative has a T_(m) of 44.9° C. Thus, the oligomer containing thephosphorothioate linkage, in combination with the bases of theinvention, is binding competent in triple helix formation.

The instant invention is shown and described herein in what isconsidered to be the most practical and preferred embodiments. It isrecognized, however, that departures can be made therefrom which arewithin the scope of the invention, and that modifications will occur tothose skilled in the art upon reading this disclosure.

1. A method of detecting the presence, absence or amount of a particularsingle-stranded DNA or RNA or a particular target duplex in a samplecomprising: selecting an oligomer having at least one base of formula(2):

wherein each X is independently O or S; R² is a group comprising atleast one pi bond connected to the carbon atom attached to the base; andPr is (H)₂ or a protecting group; and using said oligomer to detect saidDNA, RNA or target duplex.
 2. The method of 1 wherein said oligomer isused for quantitating the amount of said DNA, RNA or target duplex insaid sample.
 3. A method of performing a polymerase chain reaction (PCR)to amplify a target sequence comprising including in a PCR assay mixturean oligomer having at least one base of formula (2):

wherein each X is independently O or S; R² is a group comprising atleast one pi bond connected to the carbon atom attached to the base; andPr is (H)₂ or a protecting group; and effecting a polymerase chainreaction to amplify said target sequence.
 4. The method of claim 3further including a Taq polymerase in said PCR assay mixture.
 5. Amethod of performing a nucleic acid amplification protocol to amplify atarget nucleic acid comprising including in an assay mixture an oligomerhaving at least one base of formula (2):

wherein each X is independently O or S; R² is a group comprising atleast one pi bond connected to the carbon atom attached to Pr is (H)₂ ora protecting group; and effecting a protocol to amplify said targetnucleic acid.
 6. A method of claim 5 wherein said protocol includeshybridization of said oligomer to said target nucleic acid.